Methods and compositions for synthesis of long chain polyunsaturatd fatty acids

ABSTRACT

The present invention relates to fatty acid desaturases able to catalyze the conversion of oleic acid linoleic acid, linoleic acid to γ-linolenic acid, or of alpha-linolenic acid to stearidonic acid. Nucleic acid sequences encoding desaturases, nucleic acid sequences which hybridize thereto, DNA constructs comprising a desaturase gene, and recombinant host microorganism or animal expressing increased levels of a desaturase are described. Methods for desaturating a fatty acid and for producing a desaturated fatty acid by expressing increased levels of a desaturase are disclosed. Fatty acids, and oils containing them, which have been desaturated by a desaturase produced by recombinant host microorganisms or animals are provided. Pharmaceutical compositions, infant formulas or dietary supplements containing fatty acids which have been desaturated by a desaturase produced by a recombinant host microorganism or animal also are described.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 09/367,013 filed Aug. 5, 1999, which is a continuation-in-partapplication of U.S. patent application Ser. No. 08/834,655 filed Apr.11, 1997, both of which are incorporated by reference in theirentireties.

INTRODUCTION

1. Field of the Invention

This invention relates to modulating levels of enzymes and/or enzymecomponents relating to production of long chain poly-unsaturated fattyacids (PUFAs) in a microorganism or animal.

2. Background

Two main families of polyunsaturated fatty acids (PUFAs) are the ω3fatty acids, exemplified by eicosapentaenoic acid (EPA), and the ω6fatty acids, exemplified by arachidonic acid (ARA). PUFAs are importantcomponents of the plasma membrane of the cell, where they may be foundin such forms as phospholipids. PUFAs are necessary for properdevelopment, particularly in the developing infant brain, and for tissueformation and repair. PUFAs also serve as precursors to other moleculesof importance in human beings and animals, including the prostacyclins,eicosanoids, leukotrienes and prostaglandins. Four major long chainPUFAs of importance include docosahexaenoic acid (DHA) and EPA, whichare primarily found in different types of fish oil, γ-linolenic acid(GLA), which is found in the seeds of a number of plants, includingevening primrose (Oenothera biennis), borage (Borago officinalis) andblack currants (Ribes nigrum), and stearidonic acid (SDA), which isfound in marine oils and plant seeds. Both GLA and another importantlong chain PUFA, arachidonic acid (ARA), are found in filamentous fungi.ARA can be purified from animal tissues including liver and adrenalgland. GLA, ARA, EPA and SDA are themselves, or are dietary precursorsto, important long chain fatty acids involved in prostaglandinsynthesis, in treatment of heart disease, and in development of braintissue.

For DHA, a number of sources exist for commercial production including avariety of marine organisms, oils obtained from cold water marine fish,and egg yolk fractions. For ARA, microorganisms including the generaMortierella, Entomophthora, Phytium and Porphyridium can be used forcommercial production. Commercial sources of SDA include the generaTrichodesma and Echium. Commercial sources of GLA include eveningprimrose, black currants and borage. However, there are severaldisadvantages associated with commercial production of PUFAs fromnatural sources. Natural sources of PUFAs, such as animals and plants,tend to have highly heterogeneous oil compositions. The oils obtainedfrom these sources therefore can require extensive purification toseparate out one or more desired PUFAs or to produce an oil which isenriched in one or more PUFA. Natural sources also are subject touncontrollable fluctuations in availability. Fish stocks may undergonatural variation or may be depleted by overfishing. Fish oils haveunpleasant tastes and odors, which may be impossible to economicallyseparate from the desired product, and can render such productsunacceptable as food supplements. Animal oils, and particularly fishoils, can accumulate environmental pollutants. Weather and disease cancause fluctuation in yields from both fish and plant sources. Croplandavailable for production of alternate oil-producing crops is subject tocompetition from the steady expansion of human populations and theassociated increased need for food production on the remaining arableland. Crops which do produce PUFAs, such as borage, have not beenadapted to commercial growth and may not perform well in monoculture.Growth of such crops is thus not economically competitive where moreprofitable and better established crops can be grown. Large scalefermentation of organisms such as Mortierella is also expensive. Naturalanimal tissues contain low amounts of ARA and are difficult to process.Microorganisms such as Porphyridium and Mortierella are difficult tocultivate on a commercial scale.

Dietary supplements and pharmaceutical formulations containing PUFAs canretain the disadvantages of the PUFA source. Supplements such as fishoil capsules can contain low levels of the particular desired componentand thus require large dosages. High dosages result in ingestion of highlevels of undesired components, including contaminants. Unpleasanttastes and odors of the supplements can make such regimens undesirable,and may inhibit compliance by the patient. Care must be taken inproviding fatty acid supplements, as overaddition may result insuppression of endogenous biosynthetic pathways and lead to competitionwith other necessary fatty acids in various lipid fractions in vivo,leading to undesirable results. For example, Eskimos having a diet highin ω3 fatty acids have an increased tendency to bleed (U.S. Pat. No.4,874,603).

A number of enzymes are involved in PUFA biosynthesis. Linoleic acid(LA, 18:2 Δ9, 12) is produced from oleic acid (18:1 Δ9) by aΔ12-desaturase. GLA (18:3 Δ6, 9, 12) is produced from linoleic acid (LA,18:2 Δ9, 12) by a Δ6-desaturase. ARA (20:4 Δ5, 8, 11, 14) productionfrom dihomo-γ-linolenic acid (DGLA, 20:3 Δ8, 11, 14) is catalyzed by aΔ5-desaturase. However, animals cannot desaturate beyond the Δ9 positionand therefore cannot convert oleic acid (18:1 Δ9) into linoleic acid(18:2 Δ9, 12). Likewise, α-linolenic acid (ALA, 18:3 Δ9, 12, 15) cannotbe synthesized by mammals. Other eukaryotes, including fungi and plants,have enzymes which desaturate at positions Δ12 and Δ15. The majorpoly-unsaturated fatty acids of animals therefore are either derivedfrom diet and/or from desaturation and elongation of linoleic acid (18:2Δ9, 12) or ∝-linolenic acid (18:3 Δ9, 12, 15). Therefore it is ofinterest to obtain genetic material involved in PUFA biosynthesis fromspecies that naturally produce these fatty acids and to express theisolated material in a microbial or animal system which can bemanipulated to provide production of commercial quantities of one ormore PUFAs. Thus there is a need for fatty acid desaturases, genesencoding them, and recombinant methods of producing them. A need furtherexists for oils containing higher relative proportions of and/orenriched in specific PUFAs. A need also exists for reliable economicalmethods of producing specific PUFAs.

RELEVANT LITERATURE

Production of γ-linolenic acid by a Δ6-desaturase is described in U.S.Pat. No. 5,552,306. Production of 8,11-eicosadienoic acid usingMortierella alpina is disclosed in U.S. Pat. No. 5,376,541. Productionof docosahexaenoic acid by dinoflagellates is described in U.S. Pat. No.5,407,957. Cloning of a Δ6-palmitoyl-acyl carrier protein desaturase isdescribed in PCT publication WO 96/13591 and U.S. Pat. No. 5,614,400.Cloning of a Δ6-desaturase from borage is described in PCT publicationWO 96/21022. Cloning of Δ9-desaturases is described in the publishedpatent applications PCT WO 91/13972, EP 0 550 162 A1, EP 0 561 569 A2,EP 0 644 263 A2, and EP 0 736 598 A1, and in U.S. Pat. No. 5,057,419.Cloning of Δ12-desaturases from various organisms is described in PCTpublication WO 94/11516 and U.S. Pat. No. 5,443,974. Cloning ofΔ15-desaturases from various organisms is described in PCT publicationWO 93/11245. All publications and U.S. patents or applications referredto herein are hereby incorporated in their entirety by reference.

SUMMARY OF THE INVENTION

Novel compositions and methods are provided for preparation ofpoly-unsaturated long chain fatty acids. The compositions includenucleic acid encoding a Δ6- and Δ12-desaturase and/or polypeptideshaving Δ6- and/or Δ12-desaturase activity, the polypeptides, and probesisolating and detecting the same. The methods involve growing a hostmicroorganism or animal expressing an introduced gene or genes encodingat least one desaturase, particularly a Δ6-, Δ9-, Δ12- or Δ5-desaturase.The methods also involve the use of antisense constructs or genedisruptions to decrease or eliminate the expression level of undesireddesaturases. Regulation of expression of the desaturase polypeptide(s)provides for a relative increase in desired desaturated PUFAs as aresult of altered concentrations of enzymes and substrates involved inPUFA biosynthesis. The invention finds use, for example, in the largescale production of GLA, DGLA, ARA, EPA, DHA and SDA.

In a preferred embodiment of the invention, an isolated nucleic acidcomprising: a nucleotide sequence depicted in FIG. 3A-E (SEQ ID NO: 1)or FIG. 5A-D (SEQ ID NO: 3), a polypeptide encoded by a nucleotidesequence according FIG. 3A-E (SEQ ID NO: 1) or FIG. 5A-D (SEQ ID NO: 3),and a purified or isolated polypeptide comprising an amino acid sequencedepicted in FIG. 3A-E (SEQ ID NO: 2) or FIG. 5A-D (SEQ ID NO: 4). Inanother embodiment of the invention, provided is an isolated nucleicacid encoding a polypeptide having an amino acid sequence depicted inFIG. 3A-E (SEQ ID NO: 2) or FIG. 5A-D (SEQ ID NO: 4).

Also provided is an isolated nucleic acid comprising a nucleotidesequence which encodes a polypeptide which desaturates a fatty acidmolecule at carbon 6 or 12 from the carboxyl end, wherein saidnucleotide sequence has an average A/T content of less than about 60%.In a preferred embodiment, the isolated nucleic acid is derived from afungus, such as a fungus of the genus Mortierella. More preferred is afungus of the species Mortierella alpina.

In another preferred embodiment of the invention, an isolated nucleicacid is provided wherein the nucleotide sequence of the nucleic acid isdepicted in FIG. 3A-E (SEQ ID NO: 1) or FIG. 5A-D (SEQ ID NO: 3). Theinvention also provides an isolated or purified polypeptide whichdesaturates a fatty acid molecule at carbon 6 or 12 from the carboxylend, wherein the polypeptide is a eukaryotic polypeptide or is derivedfrom a eukaryotic polypeptide, where a preferred eukaryotic polypeptideis derived from a fungus.

The present invention further includes a nucleic acid sequence whichhybridizes to FIG. 3A-E (SEQ ID NO: 1) or FIG. 5A-D (SEQ ID NO: 3).Preferred is an isolated nucleic acid having a nucleotide sequence withat least about 50% homology to FIG. 3A-E (SEQ ID NO: 1) or FIG. 5A-D(SEQ ID NO: 3). The invention also includes an isolated nucleic acidhaving a nucleotide sequence with at least about 50% homology to FIG.3A-E (SEQ ID NO: 1) or FIG. 5A-D (SEQ ID NO: 3). In a preferredembodiment, the nucleic acid of the invention includes a nucleotidesequence which encodes an amino acid sequence depicted in FIG. 3A-D (SEQID NO: 2) which is selected from the group consisting of amino acidresidues 50-53, 39-43, 172-176, 204-213, and 390-402.

Also provided by the present invention is a nucleic acid constructcomprising a nucleotide sequence depicted in a FIG. 3A-E (SEQ ID NO: 1)or FIG. 5A-D (SEQ ID NO: 3) linked to a heterologous nucleic acid. Inanother embodiment, a nucleic acid construct is provided which comprisesa nucleotide sequence depicted in a FIG. 3A-E (SEQ ID NO: 1) or FIG.5A-D (SEQ ID NO: 3) operably associated with an expression controlsequence functional in a host cell. The host cell is either eukaryoticor prokaryotic. Preferred eukaryotic host cells are those selected fromthe group consisting of a mammalian cell, an insect cell, a fungal cell,and an algae cell. Preferred mammalian cells include an avian cell, apreferred fungal cell includes a yeast cell, and a preferred algae cellis a marine algae cell. Preferred prokaryotic cells include thoseselected from the group consisting of a bacteria, a cyanobacteria, cellswhich contain a bacteriophage, and/or a virus. The DNA sequence of therecombinant host cell preferably contains a promoter which is functionalin the host cell, which promoter is preferably inducible. In a morepreferred embodiment, the microbial cell is a fungal cell of the genusMortierella, with a more preferred fungus is of the species Mortierellaalpina.

In addition, the present invention provides a nucleic acid constructcomprising a nucleotide sequence which encodes a polypeptide comprisingan amino acid sequence which corresponds to or is complementary to anamino acid sequence depicted in FIG. 3A-E (SEQ ID NO: 2) or FIG. 5A-D(SEQ ID NO: 4), wherein the nucleic acid is operably associated with anexpression control sequence functional in a microbial cell, wherein thenucleotide sequence encodes a functionally active polypeptide whichdesaturates a fatty acid molecule at carbon 6 or carbon 12 from thecarboxyl end of a fatty acid molecule. Another embodiment of the presentinvention is a nucleic acid construct comprising a nucleotide sequencewhich encodes a functionally active Δ6-desaturase having an amino acidsequence which corresponds to or is complementary to all of or a portionof an amino acid sequence depicted in a FIG. 3A-E (SEQ ID NO: 2),wherein the nucleotide sequence is operably associated with atranscription control sequence functional in a host cell.

Yet another embodiment of the present invention is a nucleic acidconstruct comprising a nucleotide sequence which encodes a functionallyactive Δ2-desaturase having an amino acid sequence which corresponds toor is complementary to all of or a portion of an amino acid sequencedepicted in a FIG. 5A-D (SEQ ID NO: 4), wherein the nucleotide sequenceis operably associated with a transcription control sequence functionalin a host cell. The host cell, is either a eukaryotic or prokaryotichost cell. Preferred eukaryotic host cells are those selected from thegroup consisting of a mammalian cell, an insect cell, a fungal cell, andan algae cell. Preferred mammalian cells include an avian cell, apreferred fungal cell includes a yeast cell, and a preferred algae cellis a marine algae cell. Preferred prokaryotic cells include thoseselected from the group consisting of a bacteria, a cyanobacteria, cellswhich contain a bacteriophage, and/or a virus. The DNA sequence of therecombinant host cell preferably contains a promoter which is functionalin the host cell and which preferably is inducible. A preferredrecombinant host cell is a microbial cell such as a yeast cell, such asa Saccharomyces cell.

The present invention also provides a recombinant microbial cellcomprising at least one copy of a nucleic acid which encodes afunctionally active Mortierella alpina fatty acid desaturase having anamino acid sequence as depicted in FIG. 3A-E (SEQ ID NO: 2), wherein thecell or a parent of the cell was transformed with a vector comprisingsaid DNA sequence, and wherein the DNA sequence is operably associatedwith an expression control sequence. In a preferred embodiment, the cellis a microbial cell which is enriched in 18:2 fatty acids, particularlywhere the microbial cell is from a genus selected from the groupconsisting of a prokaryotic cell and eukaryotic cell. In anotherpreferred embodiment, the microbial cell according to the inventionincludes an expression control sequence which is endogenous to themicrobial cell.

Also provided by the present invention is a method for production of GLAin a host cell, where the method comprises growing a host culture havinga plurality of host cells which contain one or more nucleic acidsencoding a polypeptide which converts LA to GLA, wherein said one ormore nucleic acids is operably associated with an expression controlsequence, under conditions whereby said one or more nucleic acids areexpressed, whereby GLA is produced in the host cell. In severalpreferred embodiments of the methods, the polypeptide employed in themethod is a functionally active enzyme which desaturates a fatty acidmolecule at carbon 6 from the carboxyl end of a fatty acid molecule; thesaid one or more nucleic acids is derived from a Mortierella alpina; thesubstrate for the polypeptide is exogenously supplied; the host cellsare microbial cells; the microbial cells are yeast cells, such asSaccharomyces cells; and the growing conditions are inducible.

Also provided is an oil comprising one or more PUFA, wherein the amountof said one or more PUFAs is approximately 0.3-30% arachidonic acid(ARA), approximately 0.2-30% dihomo-γ-linolenic acid (DGLA), andapproximately 0.2-30% γ-linoleic acid (GLA). A preferred oil of theinvention is one in which the ratio of ARA:DGLA:GLA is approximately1.0:19.0:30 to 6.0:1.0:0.2. Another preferred embodiment of theinvention is a pharmaceutical composition comprising the oils in apharmaceutically acceptable carrier. Further provided is a nutritionalcomposition comprising the oils of the invention. The nutritionalcompositions of the invention preferably are administered to a mammalianhost parenterally or internally. A preferred composition of theinvention for internal consumption is an infant formula. In a preferredembodiment, the nutritional compositions of the invention are in aliquid form or a solid form, and can be formulated in or as a dietarysupplement, and the oils provided in encapsulated form. The oils of theinvention can be free of particular components of other oils and can bederived from a microbial cell, such as a yeast cell.

The present invention further provides a method for desaturating a fattyacid. In a preferred embodiment the method comprises culturing arecombinant microbial cell according to the invention under conditionssuitable for expression of a polypeptide encoded by said nucleic acid,wherein the host cell further comprises a fatty acid substrate of saidpolypeptide. Also provided is a fatty acid desaturated by such a method,and an oil composition comprising a fatty acid produced according to themethods of the invention.

The present invention further includes a purified nucleotide sequence orpolypeptide sequence that is substantially related or homologous to thenucleotide and peptide sequences presented in SEQ ID NO:1-SEQ ID NO:40.The present invention is further directed to methods of using thesequences presented in SEQ ID NO:1 to SEQ ID NO:40 as probes to identifyrelated sequences, as components of expression systems and as componentsof systems useful for producing transgenic oil.

The present invention is further directed to formulas, dietarysupplements or dietary supplements in the form of a liquid or a solidcontaining the long chain fatty acids of the invention. These formulasand supplements may be administered to a human or an animal.

The formulas and supplements of the invention may further comprise atleast one macronutrient selected from the group consisting of coconutoil, soy oil, canola oil, mono- and diglycerides, glucose, ediblelactose, electrodialysed whey, electrodialysed skim milk, milk whey, soyprotein, and other protein hydrolysates.

The formulas of the present invention may further include at least onevitamin selected from the group consisting of Vitamins A, C, D, E, and Bcomplex; and at least one mineral selected from the group consisting ofcalcium, magnesium, zinc, manganese, sodium, potassium, phosphorus,copper, chloride, iodine, selenium, and iron.

The present invention is further directed to a method of treating apatient having a condition caused by insufficient intake or productionof polyunsaturated fatty acids comprising administering to the patient adietary substitute of the invention in an amount sufficient to effecttreatment of the patient.

The present invention is further directed to cosmetic and pharmaceuticalcompositions of the material of the invention.

The present invention is further directed to transgenic oils inpharmaceutically acceptable carriers. The present invention is furtherdirected to nutritional supplements, cosmetic agents and infant formulaecontaining transgenic oils.

The present invention is further directed to a method for obtainingaltered long chain polyunsaturated fatty acid biosynthesis comprisingthe steps of: growing a microbe having cells which contain a transgenewhich encodes a transgene expression product which desaturates a fattyacid molecule at carbon 6 or 12 from the carboxyl end of said fatty acidmolecule, wherein the transgene is operably associated with anexpression control sequence, under conditions whereby the transgene isexpressed, whereby long chain polyunsaturated fatty acid biosynthesis inthe cells is altered.

The present invention is further directed toward pharmaceuticalcompositions comprising at least one nutrient selected from the groupconsisting of a vitamin, a mineral, a carbohydrate, a sugar, an aminoacid, a free fatty acid, a phospholipid, an antioxidant, and a phenoliccompound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows possible pathways for the synthesis of arachidonic acid(20:4 Δ5, 8, 11, 14) and stearidonic acid (18:4 Δ6, 9, 12, 15) frompalmitic acid (C₁₆) from a variety of organisms, including algae,Mortierella and humans. These PUFAs can serve as precursors to othermolecules important for humans and other animals, includingprostacyclins, leukotrienes, and prostaglandins, some of which areshown.

FIG. 2 shows possible pathways for production of PUFAs in addition toARA, including EPA and DHA, again compiled from a variety of organisms.

FIG. 3A-E shows the DNA sequence of the Mortierella alpina Δ6-desaturaseand the deduced amino acid sequence:

FIG. 3A-E (SEQ ID NO 1 Δ6 DESATURASE cDNA)

FIG. 3A-E (SEQ ID NO 2 Δ6 DESATURASE AMINO ACID)

FIG. 4 shows an alignment of a portion of the Mortierella alpinaΔ6-desaturase amino acid sequence with other related sequences.

FIG. 5A-D shows the DNA sequence of the Mortierella alpinaΔ12-desaturase and the deduced amino acid sequence:

FIG. 5A-D (SEQ ID NO 3 Δ12 DESATURASE cDNA)

FIG. 5A-D (SEQ ID NO 4 Δ2 DESATURASE AMINO ACID).

FIGS. 6A and 6B show the effect of different expression constructs onexpression of GLA in yeast.

FIGS. 7A and 7B show the effect of host strain on GLA production.

FIGS. 8A and 8B show the effect of temperature on GLA production in S.cerevisiae strain SC334.

FIG. 9 shows alignments of the protein sequence of the Ma 29 and contig253538a.

FIG. 10 shows alignments of the protein sequence of Ma 524 and contig253538a.

BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS

SEQ ID NO:1 shows the DNA sequence of the Mortierella alpinaΔ6-desaturase.

SEQ ID NO:2 shows the protein sequence of the Mortierella alpinaΔ6-desaturase.

SEQ ID NO:3 shows the DNA sequence of the Mortierella alpinaΔ12-desaturase.

SEQ ID NO:4 shows the protein sequence of the Mortierella alpinaΔ12-desaturase.

SEQ ID NO:5-11 show various desaturase sequences.

SEQ ID NO: 13-18 show various PCR primer sequences.

SEQ ID NO: 19 and SEQ ID NO:20 show the nucleotide and amino acidsequence of a Dictyostelium discoideum desaturase.

SEQ ID NO:21 and SEQ ID NO:22 show the nucleotide and amino acidsequence of a Phaeodactylum tricornutum desaturase.

SEQ ID NO:23-26 show the nucleotide and deduced amino acid sequence of aSchizochytrium cDNA clone.

SEQ ID NO: 27-33 show nucleotide sequences for human desaturases.

SEQ ID NO:34-SEQ ID NO:40 show peptide sequences for human desaturases.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In order to ensure a complete understanding of the invention, thefollowing definitions are provided:

Δ5-Desaturase: Δ5 desaturase is an enzyme which introduces a double bondbetween carbons 5 and 6 from the carboxyl end of a fatty acid molecule.

Δ6-Desaturase: Δ6-desaturase is an enzyme which introduces a double bondbetween carbons 6 and 7 from the carboxyl end of a fatty acid molecule.

Δ9-Desaturase: Δ9-desaturase is an enzyme which introduces a double bondbetween carbons 9 and 10 from the carboxyl end of a fatty acid molecule.

Δ2-Desaturase: Δ12-desaturase is an enzyme which introduces a doublebond between carbons 12 and 13 from the carboxyl end of a fatty acidmolecule.

Fatty Acids: Fatty acids are a class of compounds containing a longhydrocarbon chain and a terminal carboxylate group. Fatty acids includethe following: Fatty Acid 12:0 lauric acid 16:0 palmitic acid 16:1palmitoleic acid 18:0 stearic acid 18:1 oleic acid Δ9-18:1 18:2 Δ5,9taxoleic acid Δ5,9-18:2 18:2 Δ6,9 6,9-octadecadienoic acid Δ6,9-18:218:2 Linolenic acid Δ9,12-18:2 (LA) 18:3 Δ6,9,12 Gamma-linolenic acidΔ6,9,12-18:3 (GLA) 18:3 Δ5,9,12 Pinolenic acid Δ5,9,12-18:3 18:3alpha-linoleic acid Δ9,12,15-18:3 (ALA) 18:4 stearidonic acidΔ6,9,12,15-18:4 (SDA) 20:0 Arachidic acid 20:1 Eicoscenic Acid 22:0behehic acid 22:1 crucic acid 22:2 docasadienoic acid 20:4 ω6arachidonic acid Δ5,8,11,14-20:4 (ARA) 20:3 ω6 ω6-eicosatrienoicΔ8,11,14-20:3 (DGLA) dihomo-gamma linolenic 20:5 ω3 EicosapentanoicΔ5,8,11,14,17-20:5 (EPA) (Timnodonic acid) 20:3 ω3 ω3-eicosatrienoicΔ11,16,17-20:3 20:4 ω3 ω3-eicosatetraenoic Δ8,11,14,17-20:4 22:5 ω3Docosapentaenoic Δ7,10,13,16,19-22:5 (ω3 DPA) 22:6 ω3 DocosahexaenoicΔ4,7,10,13,16,19-22:6 (DHA) (cervonic acid) 24:0 Lignoceric acid

Taking into account these definitions, the present invention is directedto novel DNA sequences, DNA constructs, methods and compositions areprovided which permit modification of the poly-unsaturated long chainfatty acid content of, for example, microbial cells or animals. Hostcells are manipulated to express a sense or antisense transcript of aDNA encoding a polypeptide(s) which catalyzes the desaturation of afatty acid. The substrate(s) for the expressed enzyme may be produced bythe host cell or may be exogenously supplied. To achieve expression, thetransformed DNA is operably associated with transcriptional andtranslational initiation and termination regulatory regions that arefunctional in the host cell. Constructs comprising the gene to beexpressed can provide for integration into the genome of the host cellor can autonomously replicate in the host cell. For production oflinoleic acid (LA), the expression cassettes generally used include acassette which provides for Δ12-desaturase activity, particularly in ahost cell which produces or can take up oleic acid (U.S. Pat. No.5,443,974). Production of LA also can be increased by providing anexpression cassette for a Δ9-desaturase where that enzymatic activity islimiting. For production of ALA, the expression cassettes generally usedinclude a cassette which provides for Δ15- or ω3-desaturase activity,particularly in a host cell which produces or can take up LA. Forproduction of GLA or SDA, the expression cassettes generally usedinclude a cassette which provides for Δ6-desaturase activity,particularly in a host cell which produces or can take up LA or ALA,respectively. Production of ω6-type unsaturated fatty acids, such as LAor GLA, is favored in a host microorganism or animal which is incapableof producing ALA. The host ALA production can be removed, reduced and/orinhibited by inhibiting the activity of a Δ15- or ω3-type desaturase(see FIG. 2). This can be accomplished by standard selection, providingan expression cassette for an antisense Δ15 or ω3 transcript, bydisrupting a target Δ15- or ω3-desaturase gene through insertion,deletion, substitution of part or all of the target gene, or by addingan inhibitor of Δ15- or ω3-desaturase. Similarly, production of LA orALA is favored in a microorganism or animal having Δ6-desaturaseactivity by providing an expression cassette for an antisense Δ6transcript, by disrupting a Δ6-desaturase gene, or by use of aΔ6-desaturase inhibitor.

Microbial Production of Fatty Acids

Microbial production of fatty acids has several advantages overpurification from natural sources such as fish or plants. Many microbesare known with greatly simplified oil compositions compared with thoseof higher organisms, making purification of desired components easier.Microbial production is not subject to fluctuations caused by externalvariables such as weather and food supply. Microbially produced oil issubstantially free of contamination by environmental pollutants.Additionally, microbes can provide PUFAs in particular forms which mayhave specific uses. For example, Spirulina can provide PUFAspredominantly at the first and third positions of triglycerides;digestion by pancreatic lipases preferentially releases fatty acids fromthese positions. Following human or animal ingestion of triglyceridesderived from Spirulina, these PUFAs are released by pancreatic lipasesas free fatty acids and thus are directly available, for example, forinfant brain development. Additionally, microbial oil production can bemanipulated by controlling culture conditions, notably by providingparticular substrates for microbially expressed enzymes, or by additionof compounds which suppress undesired biochemical pathways. In additionto these advantages, production of fatty acids from recombinant microbesprovides the ability to alter the naturally occurring microbial fattyacid profile by providing new synthetic pathways in the host or bysuppressing undesired pathways, thereby increasing levels of desiredPUFAs, or conjugated forms thereof, and decreasing levels of undesiredPUFAs.

Production of Fatty Acids in Animals

Production of fatty acids in animals also presents several advantages.Expression of desaturase genes in animals can produce greatly increasedlevels of desired PUFAs in animal tissues, making recovery from thosetissues more economical. For example, where the desired PUFAs areexpressed in the breast milk of animals, methods of isolating PUFAs fromanimal milk are well established. In addition to providing a source forpurification of desired PUFAs, animal breast milk can be manipulatedthrough expression of desaturase genes, either alone or in combinationwith other human genes, to provide animal milks substantially similar tohuman breast milk during the different stages of infant development.Humanized animal milks could serve as infant formulas where humannursing is impossible or undesired, or in cases of malnourishment ordisease.

Depending upon the host cell, the availability of substrate, and thedesired end product(s), several polypeptides, particularly desaturases,are of interest. By “desaturase” is intended a polypeptide which candesaturate one or more fatty acids to produce a mono- orpoly-unsaturated fatty acid or precursor thereof of interest. Ofparticular interest are polypeptides which can catalyze the conversionof stearic acid to oleic acid, of oleic acid to LA, of LA to ALA, of LAto GLA, or of ALA to SDA, which includes enzymes which desaturate at theΔ9, Δ12, (ω6), Δ15, (ω3) or Δ6 positions. By “polypeptide” is meant anychain of amino acids, regardless of length or post-translationalmodification, for example, glycosylation or phosphorylation.Considerations for choosing a specific polypeptide having desaturaseactivity include the pH optimum of the polypeptide, whether thepolypeptide is a rate limiting enzyme or a component thereof, whetherthe desaturase used is essential for synthesis of a desiredpoly-unsaturated fatty acid, and/or co-factors required by thepolypeptide. The expressed polypeptide preferably has parameterscompatible with the biochemical environment of its location in the hostcell. For example, the polypeptide may have to compete for substratewith other enzymes in the host cell. Analyses of the K_(m) and specificactivity of the polypeptide in question therefore are considered indetermining the suitability of a given polypeptide for modifying PUFAproduction in a given host cell. The polypeptide used in a particularsituation is one which can function under the conditions present in theintended host cell but otherwise can be any polypeptide havingdesaturase activity which has the desired characteristic of beingcapable of modifying the relative production of a desired PUFA.

For production of linoleic acid from oleic acid, the DNA sequence usedencodes a polypeptide having Δ12-desaturase activity. For production ofGLA from linoleic acid, the DNA sequence used encodes a polypeptidehaving Δ6-desaturase activity. In particular instances, expression ofΔ6-desaturase activity can be coupled with expression of Δ12-desaturaseactivity and the host cell can optionally be depleted of anyΔ15-desaturase activity present, for example by providing atranscription cassette for production of antisense sequences to theΔ15-desaturase transcription product, by disrupting the Δ15-desaturasegene, or by using a host cell which naturally has, or has been mutatedto have, low Δ15-desaturase activity. Inhibition of undesired desaturasepathways also can be accomplished through the use of specific desaturaseinhibitors such as those described in U.S. Pat. No. 4,778,630. Also, ahost cell for Δ6-desaturase expression may have, or have been mutated tohave, high Δ12-desaturase activity. The choice of combination ofcassettes used depends in part on the PUFA profile and/or desaturaseprofile of the host cell. Where the host cell expresses Δ12-desaturaseactivity and lacks or is depleted in Δ15-desaturase activity,overexpression of Δ6-desaturase alone generally is sufficient to providefor enhanced GLA production. Where the host cell expresses Δ9-desaturaseactivity, expression of a Δ12- and a Δ6-desaturase can provide forenhanced GLA production. When Δ9-desaturase activity is absent orlimiting, an expression cassette for Δ9-desaturase can be used. A schemefor the synthesis of arachidonic acid (20:4 Δ^(5, 8, 11, 14)) fromstearic acid (18:0) is shown in FIG. 2. A key enzyme in this pathway isa Δ6-desaturase which converts the linoleic acid into γ-linolenic acid.Conversion of α-linolenic acid (ALA) to stearidonic acid by aΔ6-desaturase also is shown.

Sources of Polypeptides Having Desaturase Activity

A source of polypeptides having desaturase activity and oligonucleotidesencoding such polypeptides are organisms which produce a desiredpoly-unsaturated fatty acid. As an example, microorganisms having anability to produce GLA or ARA can be used as a source of Δ6- orΔ12-desaturase activity. Such microorganisms include, for example, thosebelonging to the genera Mortierella, Conidiobolus, Pythium,Phytophathora, Penicillium, Porphyridium, Coidosporium, Mucor, Fusarium,Aspergillus, Rhodotorula, and Entomophthora. Within the genusPorphyridium, of particular interest is Porphyridium cruentum. Withinthe genus Mortierella, of particular interest are Mortierella elongata,Mortierella exigua, Mortierella hygrophila, Mortierella ramanniana, var.angulispora, and Mortierella alpina. Within the genus Mucor, ofparticular interest are Mucor circinelloides and Mucor javanicus.

DNAs encoding desired desaturases can be identified in a variety ofways. As an example, a source of the desired desaturase, for examplegenomic or cDNA libraries from Mortierella, is screened with detectableenzymatically- or chemically-synthesized probes, which can be made fromDNA, RNA, or non-naturally occurring nucleotides, or mixtures thereof.Probes may be enzymatically synthesized from DNAs of known desaturasesfor normal or reduced-stringency hybridization methods. Oligonucleotideprobes also can be used to screen sources and can be based on sequencesof known desaturases, including sequences conserved among knowndesaturases, or on peptide sequences obtained from the desired purifiedprotein. Oligonucleotide probes based on amino acid sequences can bedegenerate to encompass the degeneracy of the genetic code, or can bebiased in favor of the preferred codons of the source organism.Oligonucleotides also can be used as primers for PCR from reversetranscribed mRNA from a known or suspected source; the PCR product canbe the full length cDNA or can be used to generate a probe to obtain thedesired full length cDNA. Alternatively, a desired protein can beentirely sequenced and total synthesis of a DNA encoding thatpolypeptide performed.

Once the desired genomic or cDNA has been isolated, it can be sequencedby known methods. It is recognized in the art that such methods aresubject to errors, such that multiple sequencing of the same region isroutine and is still expected to lead to measurable rates of mistakes inthe resulting deduced sequence, particularly in regions having repeateddomains, extensive secondary structure, or unusual base compositions,such as regions with high GC base content. When discrepancies arise,resequencing can be done and can employ special methods. Special methodscan include altering sequencing conditions by using: differenttemperatures; different enzymes; proteins which alter the ability ofoligonucleotides to form higher order structures; altered nucleotidessuch as ITP or methylated dGTP; different gel compositions, for exampleadding formamide; different primers or primers located at differentdistances from the problem region; or different templates such as singlestranded DNAs. Sequencing of mRNA also can be employed.

For the most part, some or all of the coding sequence for thepolypeptide having desaturase activity is from a natural source. In somesituations, however, it is desirable to modify all or a portion of thecodons, for example, to enhance expression, by employing host preferredcodons. Host preferred codons can be determined from the codons ofhighest frequency in the proteins expressed in the largest amount in aparticular host species of interest. Thus, the coding sequence for apolypeptide having desaturase activity can be synthesized in whole or inpart. All or portions of the DNA also can be synthesized to remove anydestabilizing sequences or regions of secondary structure which would bepresent in the transcribed mRNA. All or portions of the DNA also can besynthesized to alter the base composition to one more preferable in thedesired host cell. Methods for synthesizing sequences and bringingsequences together are well established in the literature. In vitromutagenesis and selection, site-directed mutagenesis, or other means canbe employed to obtain mutations of naturally occurring desaturase genesto produce a polypeptide having desaturase activity in vivo with moredesirable physical and kinetic parameters for function in the host cell,such as a longer half-life or a higher rate of production of a desiredpolyunsaturated fatty acid.

Mortierella alpina Desaturase

Of particular interest is the Mortierella alpina Δ6-desaturase, whichhas 457 amino acids and a predicted molecular weight of 51.8 kD; theamino acid sequence is shown in FIG. 3. The gene encoding theMortierella alpina Δ6-desaturase can be expressed in transgenicmicroorganisms or animals to effect greater synthesis of GLA fromlinoleic acid or of stearidonic acid from ALA. Other DNAs which aresubstantially identical to the Mortierella alpina Δ6-desaturase DNA, orwhich encode polypeptides which are substantially identical to theMortierella alpina Δ6-desaturase polypeptide, also can be used. Bysubstantially identical is intended an amino acid sequence or nucleicacid sequence exhibiting in order of increasing preference at least 60%,80%, 90% or 95% homology to the Mortierella alpina Δ6-desaturase aminoacid sequence or nucleic acid sequence encoding the amino acid sequence.For polypeptides, the length of comparison sequences generally is atleast 16 amino acids, preferably at least 20 amino acids, or mostpreferably 35 amino acids. For nucleic acids, the length of comparisonsequences generally is at least 50 nucleotides, preferably at least 60nucleotides, and more preferably at least 75 nucleotides, and mostpreferably, 110 nucleotides. Homology typically is measured usingsequence analysis software, for example, the Sequence Analysis softwarepackage of the Genetics Computer Group, University of WisconsinBiotechnology Center, 1710 University Avenue, Madison, Wis. 53705,MEGAlign (DNAStar, Inc., 1228 S. Park St., Madison, Wis. 53715), andMacVector (Oxford Molecular Group, 2105 S. Bascom Avenue, Suite 200,Campbell, Calif. 95008). Such software matches similar sequences byassigning degrees of homology to various substitutions, deletions, andother modifications. Conservative substitutions typically includesubstitutions within the following groups: glycine and alanine; valine,isoleucine and leucine; aspartic acid, glutamic acid, asparagine, andglutamine; serine and threonine; lysine and arginine; and phenylalanineand tyrosine. Substitutions may also be made on the basis of conservedhydrophobicity or hydrophilicity (Kyte and Doolittle, J. Mol. Biol. 157:105-132, 1982), or on the basis of the ability to assume similarpolypeptide secondary structure (Chou and Fasman, Adv. Enzymol. 47:45-148, 1978).

Also of interest is the Mortierella alpina Δ12-desaturase, thenucleotide and amino acid sequence of which is shown in FIG. 5. The geneencoding the Mortierella alpina Δ12-desaturase can be expressed intransgenic microorganisms or animals to effect greater synthesis of LAfrom oleic acid. Other DNAs which are substantially identical to theMortierella alpina Δ12-desaturase DNA, or which encode polypeptideswhich are substantially identical to the Mortierella alpinaΔ12-desaturase polypeptide, also can be used.

Other Desaturases

Encompassed by the present invention are related desaturases from thesame or other organisms. Such related desaturases include variants ofthe disclosed Δ6- or Δ12-desaturase naturally occurring within the sameor different species of Mortierella, as well as homologues of thedisclosed Δ6- or Δ12-desaturase from other species. Also included aredesaturases which, although not substantially identical to theMortierella alpina Δ6- or Δ12-desaturase, desaturate a fatty acidmolecule at carbon 6 or 12, respectively, from the carboxyl end of afatty acid molecule, or at carbon 12 or 6 from the terminal methylcarbon in an 18 carbon fatty acid molecule. Related desaturases can beidentified by their ability to function substantially the same as thedisclosed desaturases; that is, are still able to effectively convert LAto GLA, ALA to SDA or oleic acid to LA. Related desaturases also can beidentified by screening sequence databases for sequences homologous tothe disclosed desaturases, by hybridization of a probe based on thedisclosed desaturases to a library constructed from the source organism,or by RT-PCR using mRNA from the source organism and primers based onthe disclosed desaturases. Such desaturases include those from humans,Dictyostelium discoideum and Phaeodactylum tricornum.

The regions of a desaturase polypeptide important for desaturaseactivity can be determined through routine mutagenesis, expression ofthe resulting mutant polypeptides and determination of their activities.Mutants may include deletions, insertions and point mutations, orcombinations thereof. A typical functional analysis begins with deletionmutagenesis to determine the N- and C-terminal limits of the proteinnecessary for function, and then internal deletions, insertions or pointmutants are made to further determine regions necessary for function.Other techniques such as cassette mutagenesis or total synthesis alsocan be used. Deletion mutagenesis is accomplished, for example, by usingexonucleases to sequentially remove the 5′ or 3′ coding regions. Kitsare available for such techniques. After deletion, the coding region iscompleted by ligating oligonucleotides containing start or stop codonsto the deleted coding region after 5′ or 3′ deletion, respectively.Alternatively, oligonucleotides encoding start or stop codons areinserted into the coding region by a variety of methods includingsite-directed mutagenesis, mutagenic PCR or by ligation onto DNAdigested at existing restriction sites. Internal deletions can similarlybe made through a variety of methods including the use of existingrestriction sites in the DNA, by use of mutagenic primers via sitedirected mutagenesis or mutagenic PCR. Insertions are made throughmethods such as linker-scanning mutagenesis, site-directed mutagenesisor mutagenic PCR. Point mutations are made through techniques such assite-directed mutagenesis or mutagenic PCR.

Chemical mutagenesis also can be used for identifying regions of adesaturase polypeptide important for activity. A mutated construct isexpressed, and the ability of the resulting altered protein to functionas a desaturase is assayed. Such structure-function analysis candetermine which regions may be deleted, which regions tolerateinsertions, and which point mutations allow the mutant protein tofunction in substantially the same way as the native desaturase. Allsuch mutant proteins and nucleotide sequences encoding them are withinthe scope of the present invention.

Expression of Desaturase Genes

Once the DNA encoding a desaturase polypeptide has been obtained, it isplaced in a vector capable of replication in a host cell, or ispropagated in vitro by means of techniques such as PCR or long PCR.Replicating vectors can include plasmids, phage, viruses, cosmids andthe like. Desirable vectors include those useful for mutagenesis of thegene of interest or for expression of the gene of interest in hostcells. The technique of long PCR has made in vitro propagation of largeconstructs possible, so that modifications to the gene of interest, suchas mutagenesis or addition of expression signals, and propagation of theresulting constructs can occur entirely in vitro without the use of areplicating vector or a host cell.

For expression of a desaturase polypeptide, functional transcriptionaland translational initiation and termination regions are operably linkedto the DNA encoding the desaturase polypeptide. Expression of thepolypeptide coding region can take place in vitro or in a host cell.Transcriptional and translational initiation and termination regions arederived from a variety of nonexclusive sources, including the DNA to beexpressed, genes known or suspected to be capable of expression in thedesired system, expression vectors, chemical synthesis, or from anendogenous locus in a host cell.

Expression In Vitro

In vitro expression can be accomplished, for example, by placing thecoding region for the desaturase polypeptide in an expression vectordesigned for in vitro use and adding rabbit reticulocyte lysate andcofactors; labeled amino acids can be incorporated if desired. Such invitro expression vectors may provide some or all of the expressionsignals necessary in the system used. These methods are well known inthe art and the components of the system are commercially available. Thereaction mixture can then be assayed directly for the polypeptide, forexample by determining its activity, or the synthesized polypeptide canbe purified and then assayed.

Expression in a Host Cell

Expression in a host cell can be accomplished in a transient or stablefashion. Transient expression can occur from introduced constructs whichcontain expression signals functional in the host cell, but whichconstructs do not replicate and rarely integrate in the host cell, orwhere the host cell is not proliferating. Transient expression also canbe accomplished by inducing the activity of a regulatable promoteroperably linked to the gene of interest, although such inducible systemsfrequently exhibit a low basal level of expression. Stable expressioncan be achieved by introduction of a construct that can integrate intothe host genome or that autonomously replicates in the host cell. Stableexpression of the gene of interest can be selected for through the useof a selectable marker located on or transfected with the expressionconstruct, followed by selection for cells expressing the marker. Whenstable expression results from integration, integration of constructscan occur randomly within the host genome or can be targeted through theuse of constructs containing regions of homology with the host genomesufficient to target recombination with the host locus. Where constructsare targeted to an endogenous locus, all or some of the transcriptionaland translational regulatory regions can be provided by the endogenouslocus.

When increased expression of the desaturase polypeptide in the sourceorganism is desired, several methods can be employed. Additional genesencoding the desaturase polypeptide can be introduced into the hostorganism. Expression from the native desaturase locus also can beincreased through homologous recombination, for example by inserting astronger promoter into the host genome to cause increased expression, byremoving destabilizing sequences from either the mRNA or the encodedprotein by deleting that information from the host genome, or by addingstabilizing sequences to the mRNA (U.S. Pat. No. 4,910,141).

When it is desirable to express more than one different gene,appropriate regulatory regions and expression methods, introduced genescan be propagated in the host cell through use of replicating vectors orby integration into the host genome. Where two or more genes areexpressed from separate replicating vectors, it is desirable that eachvector has a different means of replication. Each introduced construct,whether integrated or not, should have a different means of selectionand should lack homology to the other constructs to maintain stableexpression and prevent reassortment of elements among constructs.Judicious choices of regulatory regions, selection means and method ofpropagation of the introduced construct can be experimentally determinedso that all introduced genes are expressed at the necessary levels toprovide for synthesis of the desired products.

As an example, where the host cell is a yeast, transcriptional andtranslational regions functional in yeast cells are provided,particularly from the host species. The transcriptional initiationregulatory regions can be obtained, for example from genes in theglycolytic pathway, such as alcohol dehydrogenase,glyceraldehyde-3-phosphate dehydrogenase (GPD), phosphoglucoisomerase,phosphoglycerate kinase, etc. or regulatable genes such as acidphosphatase, lactase, metallothionein, glucoamylase, etc. Any one of anumber of regulatory sequences can be used in a particular situation,depending upon whether constitutive or induced transcription is desired,the particular efficiency of the promoter in conjunction with theopen-reading frame of interest, the ability to join a strong promoterwith a control region from a different promoter which allows forinducible transcription, ease of construction, and the like. Ofparticular interest are promoters which are activated in the presence ofgalactose. Galactose-inducible promoters (GAL1, GAL7, and GAL10) havebeen extensively utilized for high level and regulated expression ofprotein in yeast (Lue et al., Mol. Cell. Biol. Vol. 7, p. 3446, 1987;Johnston, Microbiol. Rev. Vol. 51, p. 458, 1987). Transcription from theGAL promoters is activated by the GAL4 protein, which binds to thepromoter region and activates transcription when galactose is present.In the absence of galactose, the antagonist GAL80 binds to GAL4 andprevents GAL4 from activating transcription. Addition of galactoseprevents GAL80 from inhibiting activation by GAL4.

Nucleotide sequences surrounding the translational initiation codon ATGhave been found to affect expression in yeast cells. If the desiredpolypeptide is poorly expressed in yeast, the nucleotide sequences ofexogenous genes can be modified to include an efficient yeasttranslation initiation sequence to obtain optimal gene expression. Forexpression in Saccharomyces, this can be done by site-directedmutagenesis of an inefficiently expressed gene by fusing it in-frame toan endogenous Saccharomyces gene, preferably a highly expressed gene,such as the lactase gene.

The termination region can be derived from the 3′ region of the genefrom which the initiation region was obtained or from a different gene.A large number of termination regions are known to and have been foundto be satisfactory in a variety of hosts from the same and differentgenera and species. The termination region usually is selected more as amatter of convenience rather than because of any particular property.Preferably, the termination region is derived from a yeast gene,particularly Saccharomyces, Schizosaccharomyces, Candida orKluyveromyces. The 3′ regions of two mammalian genes, γ interferon andα2 interferon, are also known to function in yeast.

Introduction of Constructs into Host Cells

Constructs comprising the gene of interest may be introduced into a hostcell by standard techniques. These techniques include transformation,protoplast fusion, lipofection, transfection, transduction, conjugation,infection, bolistic impact, electroporation, microinjection, scraping,or any other method which introduces the gene of interest into the hostcell. Methods of transformation which are used include lithium acetatetransformation (Methods in Enzymology, Vol. 194, p. 186-187, 1991). Forconvenience, a host cell which has been manipulated by any method totake up a DNA sequence or construct will be referred to as “transformed”or “recombinant” herein.

The subject host will have at least have one copy of the expressionconstruct and may have two or more, depending upon whether the gene isintegrated into the genome, amplified, or is present on anextrachromosomal element having multiple copy numbers. Where the subjecthost is a yeast, four principal types of yeast plasmid vectors can beused: Yeast Integrating plasmids (YIps), Yeast Replicating plasmids(YRps), Yeast Centromere plasmids (YCps), and Yeast Episomal plasmids(YEps). YIps lack a yeast replication origin and must be propagated asintegrated elements in the yeast genome. YRps have a chromosomallyderived autonomously replicating sequence and are propagated as mediumcopy number (20 to 40), autonomously replicating, unstably segregatingplasmids. YCps have both a replication origin and a centromere sequenceand propagate as low copy number (10-20), autonomously replicating,stably segregating plasmids. YEps have an origin of replication from theyeast 2 μm plasmid and are propagated as high copy number, autonomouslyreplicating, irregularly segregating plasmids. The presence of theplasmids in yeast can be ensured by maintaining selection for a markeron the plasmid. Of particular interest are the yeast vectors pYES2 (aYEp plasmid available from Invitrogen, confers uracil prototrophy and aGAL1 galactose-inducible promoter for expression), pRS425-pG1 (a YEpplasmid obtained from Dr. T. H. Chang, Ass. Professor of MolecularGenetics, Ohio State University, containing a constitutive GPD promoterand conferring leucine prototrophy), and pYX424 (a YEp plasmid having aconstitutive TP1 promoter and conferring leucine prototrophy; Alber, T.and Kawasaki, G. (1982). J. Mol. & Appl. Genetics 1: 419).

The transformed host cell can be identified by selection for a markercontained on the introduced construct. Alternatively, a separate markerconstruct may be introduced with the desired construct, as manytransformation techniques introduce many DNA molecules into host cells.Typically, transformed hosts are selected for their ability to grow onselective media. Selective media may incorporate an antibiotic or lack afactor necessary for growth of the untransformed host, such as anutrient or growth factor. An introduced marker gene therefor may conferantibiotic resistance, or encode an essential growth factor or enzyme,and permit growth on selective media when expressed in the transformedhost. Selection of a transformed host can also occur when the expressedmarker protein can be detected, either directly or indirectly. Themarker protein may be expressed alone or as a fusion to another protein.The marker protein can be detected by its enzymatic activity; forexample β galactosidase can convert the substrate X-gal to a coloredproduct, and luciferase can convert luciferin to a light-emittingproduct. The marker protein can be detected by its light-producing ormodifying characteristics; for example, the green fluorescent protein ofAequorea victoria fluoresces when illuminated with blue light.Antibodies can be used to detect the marker protein or a molecular tagon, for example, a protein of interest. Cells expressing the markerprotein or tag can be selected, for example, visually, or by techniquessuch as FACS or panning using antibodies. For selection of yeasttransformants, any marker that functions in yeast may be used.Desirably, resistance to kanamycin and the amino glycoside G418 are ofinterest, as well as ability to grow on media lacking uracil, leucine,lysine or tryptophan.

Of particular interest is the Δ6- and Δ12-desaturase-mediated productionof PUFAs in prokaryotic and eukaryotic host cells. Prokaryotic cells ofinterest include Eschericia, Bacillus, Lactobacillus, cyanobacteria andthe like. Eukaryotic cells include mammalian cells such as those oflactating animals, avian cells such as of chickens, and other cellsamenable to genetic manipulation including insect, fungal, and algaecells. The cells may be cultured or formed as part or all of a hostorganism including an animal. Viruses and bacteriophage also may be usedwith the cells in the production of PUFAs, particularly for genetransfer, cellular targeting and selection. In a preferred embodiment,the host is any microorganism or animal which produces and/or canassimilate exogenously supplied substrate(s) for a Δ6- and/orΔ12-desaturase, and preferably produces large amounts of one or more ofthe substrates. Examples of host animals include mice, rats, rabbits,chickens, quail, turkeys, bovines, sheep, pigs, goats, yaks, etc., whichare amenable to genetic manipulation and cloning for rapid expansion ofthe transgene expressing population. For animals, the desaturasetransgene(s) can be adapted for expression in target organelles, tissuesand body fluids through modification of the gene regulatory regions. Ofparticular interest is the production of PUFAs in the breast milk of thehost animal.

Expression in Yeast

Examples of host microorganisms include Saccharomyces cerevisiae,Saccharomyces carlsbergensis, or other yeast such as Candida,Kluyveromyces or other fungi, for example, filamentous fungi such asAspergillus, Neurospora, Penicillium, etc. Desirable characteristics ofa host microorganism are, for example, that it is genetically wellcharacterized, can be used for high level expression of the productusing ultra-high density fermentation, and is on the GRAS (generallyrecognized as safe) list since the proposed end product is intended foringestion by humans. Of particular interest is use of a yeast, moreparticularly baker's yeast (S. cerevisiae), as a cell host in thesubject invention. Strains of particular interest are SC334 (Mat αpep4-3 prb1-1122 ura3-52 leu2-3, 112 reg1-501 gal1; Gene 83:57-64, 1989,Hovland P. et al.), YTC34 (α ade2-101 his3Δ200 lys2-801 ura3-52;obtained from Dr. T. H. Chang, Ass. Professor of Molecular Genetics,Ohio State University), YTC41 (a/α ura3-52/ura3=52 lys2-801/lys2-801ade2-101/ade2-101 trp1-Δ1/trp1-Δ1 his3Δ200/his3Δ200 leu2Δ1/leu2Δ1;obtained from Dr. T. H. Chang, Ass. Professor of Molecular Genetics,Ohio State University), BJ1995 (obtained from the Yeast Genetic StockCentre, 1021 Donner Laboratory, Berkeley, Calif. 94720), INVSC1 (Mat αhiw3Δ1 leu2 trp1-289 ura3-52; obtained from Invitrogen, 1600 FaradayAve., Carlsbad, Calif. 92008) and INVSC2 (Mat α his3Δ200 ura3-167;obtained from Invitrogen).

Expression in Avian Species

For producing PUFAs in avian species and cells, such as chickens,turkeys, quail and ducks, gene transfer can be performed by introducinga nucleic acid sequence encoding a Δ6 and/or Δ12-desaturase into thecells following procedures known in the art. If a transgenic animal isdesired, pluripotent stem cells of embryos can be provided with a vectorcarrying a desaturase encoding transgene and developed into adult animal(U.S. Pat. No. 5,162,215; Ono et al. (1996) Comparative Biochemistry andPhysiology A 113(3):287-292; WO 9612793; WO 9606160). In most cases, thetransgene will be modified to express high levels of the desaturase inorder to increase production of PUFAs. The transgene can be modified,for example, by providing transcriptional and/or translationalregulatory regions that function in avian cells, such as promoters whichdirect expression in particular tissues and egg parts such as yolk. Thegene regulatory regions can be obtained from a variety of sources,including chicken anemia or avian leukosis viruses or avian genes suchas a chicken ovalbumin gene.

Expression in Insect Cells

Production of PUFAs in insect cells can be conducted using baculovirusexpression vectors harboring one or more desaturase transgenes.Baculovirus expression vectors are available from several commercialsources such as Clonetech. Methods for producing hybrid and transgenicstrains of algae, such as marine algae, which contain and express adesaturase transgene also are provided. For example, transgenic marinealgae may be prepared as described in U.S. Pat. No. 5,426,040. As withthe other expression systems described above, the timing, extent ofexpression and activity of the desaturase transgene can be regulated byfitting the polypeptide coding sequence with the appropriatetranscriptional and translational regulatory regions selected for aparticular use. Of particular interest are promoter regions which can beinduced under preselected growth conditions. For example, introductionof temperature sensitive and/or metabolite responsive mutations into thedesaturase transgene coding sequences, its regulatory regions, and/orthe genome of cells into which the transgene is introduced can be usedfor this purpose.

The transformed host cell is grown under appropriate conditions adaptedfor a desired end result. For host cells grown in culture, theconditions are typically optimized to produce the greatest or mosteconomical yield of PUFAs, which relates to the selected desaturaseactivity. Media conditions which may be optimized include: carbonsource, nitrogen source, addition of substrate, final concentration ofadded substrate, form of substrate added, aerobic or anaerobic growth,growth temperature, inducing agent, induction temperature, growth phaseat induction, growth phase at harvest, pH, density, and maintenance ofselection. Microorganisms of interest, such as yeast are preferablygrown in selected medium. For yeast, complex media such as peptone broth(YPD) or a defined media such as a minimal media (contains amino acids,yeast nitrogen base, and ammonium sulfate, and lacks a component forselection, for example uracil) are preferred. Desirably, substrates tobe added are first dissolved in ethanol. Where necessary, expression ofthe polypeptide of interest may be induced, for example by including oradding galactose to induce expression from a GAL promoter.

Expression in Plants

Production of PUFA's in plants can be conducted using various planttransformation systems such as the use of Agrobacterium tumefaciens,plant viruses, particle cell transformation and the like which aredisclosed in Applicant's related applications U.S. application Ser. Nos.08/834,033 and 08/956,985 and continuation-in-part applications filedsimultaneously with this application all of which are herebyincorporated by reference.

Expression in an Animal

Expression in cells of a host animal can likewise be accomplished in atransient or stable manner. Transient expression can be accomplished viaknown methods, for example infection or lipofection, and can be repeatedin order to maintain desired expression levels of the introducedconstruct (see Ebert, PCT publication WO 94/05782). Stable expressioncan be accomplished via integration of a construct into the host genome,resulting in a transgenic animal. The construct can be introduced, forexample, by microinjection of the construct into the pronuclei of afertilized egg, or by transfection, retroviral infection or othertechniques whereby the construct is introduced into a cell line whichmay form or be incorporated into an adult animal (U.S. Pat. No.4,873,191; U.S. Pat. No. 5,530,177; U.S. Pat. No. 5,565,362; U.S. Pat.No. 5,366,894; Willmut et al (1997) Nature 385:810). The recombinanteggs or embryos are transferred to a surrogate mother (U.S. Pat. No.4,873,191; U.S. Pat. No. 5,530,177; U.S. Pat. No. 5,565,362; U.S. Pat.No. 5,366,894; Wilmut et al (supra)).

After birth, transgenic animals are identified, for example, by thepresence of an introduced marker gene, such as for coat color, or by PCRor Southern blotting from a blood, milk or tissue sample to detect theintroduced construct, or by an immunological or enzymological assay todetect the expressed protein or the products produced therefrom (U.S.Pat. No. 4,873,191; U.S. Pat. No. 5,530,177; U.S. Pat. No. 5,565,362;U.S. Pat. No. 5,366,894; Wilmut et al (supra)). The resulting transgenicanimals may be entirely transgenic or may be mosaics, having thetransgenes in only a subset of their cells. The advent of mammaliancloning, accomplished by fusing a nucleated cell with an enucleated egg,followed by transfer into a surrogate mother, presents the possibilityof rapid, large-scale production upon obtaining a “founder” animal orcell comprising the introduced construct; prior to this, it wasnecessary for the transgene to be present in the germ line of the animalfor propagation (Wilmut et al (supra)).

Expression in a host animal presents certain efficiencies, particularlywhere the host is a domesticated animal. For production of PUFAs in afluid readily obtainable from the host animal, such as milk, thedesaturase transgene can be expressed in mammary cells from a femalehost, and the PUFA content of the host cells altered. The desaturasetransgene can be adapted for expression so that it is retained in themammary cells, or secreted into milk, to form the PUFA reaction productslocalized to the milk (PCT publication WO 95/24488). Expression can betargeted for expression in mammary tissue using specific regulatorysequences, such as those of bovine α-lactalbumin, α-casein, β-casein,γ-casein, κ-casein, β-lactoglobulin, or whey acidic protein, and mayoptionally include one or more introns and/or secretory signal sequences(U.S. Pat. No. 5,530,177; Rosen, U.S. Pat. No. 5,565,362; Clark et al.,U.S. Pat. No. 5,366,894; Garner et al., PCT publication WO 95/23868).Expression of desaturase transgenes, or antisense desaturasetranscripts, adapted in this manner can be used to alter the levels ofspecific PUFAs, or derivatives thereof, found in the animals milk.Additionally, the desaturase transgene(s) can be expressed either byitself or with other transgenes, in order to produce animal milkcontaining higher proportions of desired PUFAs or PUFA ratios andconcentrations that resemble human breast milk (Prieto et al., PCTpublication WO 95/24494).

Purification of Fatty Acids

The desaturated fatty acids may be found in the host microorganism oranimal as free fatty acids or in conjugated forms such as acylglycerols,phospholipids, sulfolipids or glycolipids, and may be extracted from thehost cell through a variety of means well-known in the art. Such meansmay include extraction with organic solvents, sonication, supercriticalfluid extraction using for example carbon dioxide, and physical meanssuch as presses, or combinations thereof. Of particular interest isextraction with hexane or methanol and chloroform. Where desirable, theaqueous layer can be acidified to protonate negatively charged moietiesand thereby increase partitioning of desired products into the organiclayer. After extraction, the organic solvents can be removed byevaporation under a stream of nitrogen. When isolated in conjugatedforms, the products may be enzymatically or chemically cleaved torelease the free fatty acid or a less complex conjugate of interest, andcan then be subject to further manipulations to produce a desired endproduct. Desirably, conjugated forms of fatty acids are cleaved withpotassium hydroxide.

If further purification is necessary, standard methods can be employed.Such methods may include extraction, treatment with urea, fractionalcrystallization, HPLC, fractional distillation, silica gelchromatography, high speed centrifugation or distillation, orcombinations of these techniques. Protection of reactive groups, such asthe acid or alkenyl groups, may be done at any step through knowntechniques, for example alkylation or iodination. Methods used includemethylation of the fatty acids to produce methyl esters. Similarly,protecting groups may be removed at any step. Desirably, purification offractions containing GLA, SDA, ARA, DHA and EPA may be accomplished bytreatment with urea and/or fractional distillation.

Uses of Fatty Acids

The fatty acids of the subject invention finds many applications. Probesbased on the DNAs of the present invention may find use in methods forisolating related molecules or in methods to detect organisms expressingdesaturases. When used as probes, the DNAs or oligonucleotides must bedetectable. This is usually accomplished by attaching a label either atan internal site, for example via incorporation of a modified residue,or at the 5′ or 3′ terminus. Such labels can be directly detectable, canbind to a secondary molecule that is detectably labeled, or can bind toan unlabelled secondary molecule and a detectably labeled tertiarymolecule; this process can be extended as long as is practical toachieve a satisfactorily detectable signal without unacceptable levelsof background signal. Secondary, tertiary, or bridging systems caninclude use of antibodies directed against any other molecule, includinglabels or other antibodies, or can involve any molecules which bind toeach other, for example a biotin-streptavidin/avidin system. Detectablelabels typically include radioactive isotopes, molecules whichchemically or enzymatically produce or alter light, enzymes whichproduce detectable reaction products, magnetic molecules, fluorescentmolecules or molecules whose fluorescence or light-emittingcharacteristics change upon binding. Examples of labelling methods canbe found in U.S. Pat. No. 5,011,770. Alternatively, the binding oftarget molecules can be directly detected by measuring the change inheat of solution on binding of probe to target via isothermal titrationcalorimetry, or by coating the probe or target on a surface anddetecting the change in scattering of light from the surface produced bybinding of target or probe, respectively, as may be done with theBIAcore system.

PUFAs produced by recombinant means find applications in a wide varietyof areas. Supplementation of animals or humans with PUFAs in variousforms can result in increased levels not only of the added PUFAs but oftheir metabolic progeny as well.

Nutritional Compositions

The present invention also includes nutritional compositions. Suchcompositions, for purposes of the present invention, include any food orpreparation for human consumption including for enteral or parenteralconsumption, which when taken into the body (a) serve to nourish orbuild up tissues or supply energy and/or (b) maintain, restore orsupport adequate nutritional status or metabolic function.

The nutritional composition of the present invention comprises at leastone oil or acid produced in accordance with the present invention andmay either be in a solid or liquid form. Additionally, the compositionmay include edible macronutrients, vitamins and minerals in amountsdesired for a particular use. The amount of such ingredients will varydepending on whether the composition is intended for use with normal,healthy infants, children or adults having specialized needs such asthose which accompany certain metabolic conditions (e.g., metabolicdisorders).

Examples of macronutrients which may be added to the composition includebut are not limited to edible fats, carbohydrates and proteins. Examplesof such edible fats include but are not limited to coconut oil, soy oil,and mono- and diglycerides. Examples of such carbohydrates include butare not limited to glucose, edible lactose and hydrolyzed search.Additionally, examples of proteins which may be utilized in thenutritional composition of the invention include but are not limited tosoy proteins, electrodialysed whey, electrodialysed skim milk, milkwhey, or the hydrolysates of these proteins.

With respect to vitamins and minerals, the following may be added to thenutritional compositions of the present invention: calcium, phosphorus,potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc,selenium, iodine, and Vitamins A, E, D, C, and the B complex. Other suchvitamins and minerals may also be added.

The components utilized in the nutritional compositions of the presentinvention will of semi-purified or purified origin. By semi-purified orpurified is meant a material which has been prepared by purification ofa natural material or by synthesis.

Examples of nutritional compositions of the present invention includebut are not limited to infant formulas, dietary supplements, andrehydration compositions. Nutritional compositions of particularinterest include but are not limited to those utilized for enteral andparenteral supplementation for infants, specialist infant formulae,supplements for the elderly, and supplements for those withgastrointestinal difficulties and/or malabsorption.

Nutritional Compositions

A typical nutritional composition of the present invention will containedible macronutrients, vitamins and minerals in amounts desired for aparticular use. The amounts of such ingredients will vary depending onwhether the formulation is intended for use with normal, healthyindividuals temporarily exposed to stress, or to subjects havingspecialized needs due to certain chronic or acute disease states (e.g.,metabolic disorders). It will be understood by persons skilled in theart that the components utilized in a nutritional formulation of thepresent invention are of semi-purified or purified origin. Bysemi-purified or purified is meant a material that has been prepared bypurification of a natural material or by synthesis. These techniques arewell known in the art (See, e.g., Code of Federal Regulations for FoodIngredients and Food Processing; Recommended Dietary Allowances, 10^(th)Ed., National Academy Press, Washington, D.C., 1989).

In a preferred embodiment, a nutritional formulation of the presentinvention is an enteral nutritional product, more preferably an adult orchild enteral nutritional product. Accordingly in a further aspect ofthe invention, a nutritional formulation is provided that is suitablefor feeding adults or children, who are experiencing stress. The formulacomprises, in addition to the PUFAs of the invention; macronutrients,vitamins and minerals in amounts designed to provide the dailynutritional requirements of adults.

The macronutritional components include edible fats, carbohydrates andproteins. Exemplary edible fats are coconut oil, soy oil, and mono- anddiglycerides and the PUFA oils of this invention. Exemplarycarbohydrates are glucose, edible lactose and hydrolyzed cornstarch. Atypical protein source would be soy protein, electrodialysed whey orelectrodialysed skim milk or milk whey, or the hydrolysates of theseproteins, although other protein sources are also available and may beused. These macronutrients would be added in the form of commonlyaccepted nutritional compounds in amount equivalent to those present inhuman milk or an energy basis, i.e., on a per calorie basis.

Methods for formulating liquid and enteral nutritional formulas are wellknown in the art and are described in detail in the examples.

The enteral formula can be sterilized and subsequently utilized on aready-to-feed (RTF) basis or stored in a concentrated liquid or apowder. The powder can be prepared by spray drying the enteral formulaprepared as indicated above, and the formula can be reconstituted byrehydrating the concentrate. Adult and infant nutritional formulas arewell known in the art and commercially available (e.g., Similac®,Ensure®, Jevity® and Alimentum® from Ross Products Division, AbbottLaboratories). An oil or acid of the present invention can be added toany of these formulas in the amounts described below.

The energy density of the nutritional composition when in liquid form,can typically range from about 0.6 to 3.0 Kcal per ml. When in solid orpowdered form, the nutritional supplement can contain from about 1.2 tomore than 9 Kcals per gm, preferably 3 to 7 Kcals per gm. In general,the osmolality of a liquid product should be less than 700 mOsm and morepreferably less than 660 mOsm.

The nutritional formula would typically include vitamins and minerals,in addition to the PUFAs of the invention, in order to help theindividual ingest the minimum daily requirements for these substances.In addition to the PUFAs listed above, it may also be desirable tosupplement the nutritional composition with zinc, copper, and folic acidin addition to antioxidants. It is believed that these substances willalso provide a boost to the stressed immune system and thus will providefurther benefits to the individual. The presence of zinc, copper orfolic acid is optional and is not required in order to gain thebeneficial effects on immune suppression. Likewise a pharmaceuticalcomposition can be supplemented with these same substances as well.

In a more preferred embodiment, the nutritional contains, in addition tothe antioxidant system and the PUFA component, a source of carbohydratewherein at least 5 weight % of said carbohydrate is an indigestibleoligosaccharide. In yet a more preferred embodiment, the nutritionalcomposition additionally contains protein, taurine and carnitine.

The PUFAs, or derivatives thereof made by the disclosed method can beused as dietary substitutes, or supplements, particularly infantformulas, for patients undergoing intravenous feeding or for preventingor treating malnutrition. Typically, human breast milk has a fatty acidprofile comprising from about 0.15% to about 0.36% as DHA, from about0.03% to about 0.13% as EPA, from about 0.30% to about 0.88% as ARA,from about 0.22% to about 0.67% as DGLA, and from about 0.27% to about1.04% as GLA. Additionally, the predominant triglyceride in human milkhas been reported to be 1,3-di-oleoyl-2-palmitoyl, with 2-palmitoylglycerides reported as better absorbed than 2-oleoyl or 2-lineoylglycerides (U.S. Pat. No. 4,876,107). Thus, fatty acids such as ARA,DGLA, GLA and/or EPA produced by the invention can be used to alter thecomposition of infant formulas to better replicate the PUFA compositionof human breast milk. In particular, an oil composition for use in apharmacologic or food supplement, particularly a breast milk substituteor supplement, will preferably comprise one or more of ARA, DGLA andGLA. More preferably the oil will comprise from about 0.3 to 30% ARA,from about 0.2 to 30% DGLA, and from about 0.2 to about 30% GLA.

In addition to the concentration, the ratios of ARA, DGLA and GLA can beadapted for a particular given end use. When formulated as a breast milksupplement or substitute, an oil composition which contains two or moreof ARA, DGLA and GLA will be provided in a ratio of about 1:19:30 toabout 6:1:0.2, respectively. For example, the breast milk of animals canvary in ratios of ARA:DGLA:DGL ranging from 1:19:30 to 6:1:0.2, whichincludes intermediate ratios which are preferably about 1:1:1, 1:2:1,1:1:4. When produced together in a host cell, adjusting the rate andpercent of conversion of a precursor substrate such as GLA and DGLA toARA can be used to precisely control the PUFA ratios. For example, a 5%to 10% conversion rate of DGLA to ARA can be used to produce an ARA toDGLA ratio of about 1:19, whereas a conversion rate of about 75% to 80%can be used to produce an ARA to DGLA ratio of about 6:1. Therefore,whether in a cell culture system or in a host animal, regulating thetiming, extent and specificity of desaturase expression as described canbe used to modulate the PUFA levels and ratios. Depending on theexpression system used, e.g., cell culture or an animal expressingoil(s) in its milk, the oils also can be isolated and recombined in thedesired concentrations and ratios. Amounts of oils providing theseratios of PUFA can be determined following standard protocols. PUFAs, orhost cells containing them, also can be used as animal food supplementsto alter an animal's tissue or milk fatty acid composition to one moredesirable for human or animal consumption.

For dietary supplementation, the purified PUFAs, or derivatives thereofmay be incorporated into cooking oils, fats or margarines formulated sothat in normal use the recipient would receive the desired amount. ThePUFAs may also be incorporated into infant formulas, nutritionalsupplements or other food products, and may find use asanti-inflammatory or cholesterol lowering agents.

Pharmaceutical Compositions

The present invention also encompasses a pharmaceutical compositioncomprising one or more of the acids and/or resulting oils produced inaccordance with the methods described herein. More specifically, such apharmaceutical composition may comprise one or more of the acids and/oroils as well as a standard, well-known, non-toxic pharmaceuticallyacceptable carrier, adjuvant or vehicle such as, for example, phosphatebuffered saline, water, ethanol, polyols, vegetable oils, a wettingagent or an emulsion such as a water/oil emulsion. The composition maybe in either a liquid or solid form. For example, the composition may bein the form of a tablet, capsule, ingestible liquid or powder,injectible, or topical ointment or cream.

Possible routes of administration include, for example, oral, rectal andparenteral. The route of administration will, of course, depend upon thedesired effect. For example, if the composition is being utilized totreat rough, dry, or aging skin, to treat injured or burned skin, or totreat skin or hair affected by a disease or condition, it may perhaps beapplied topically.

The dosage of the composition to be administered to the patient may bedetermined by one of ordinary skill in the art and depends upon variousfactors such as weight of the patient, age of the patient, immune statusof the patient, etc.

With respect to form, the composition may be, for example, a solution, adispersion, a suspension, an emulsion or a sterile powder which is thenreconstituted.

Additionally, the composition of the present invention may be utilizedfor cosmetic purposes. It may be added to pre-existing cosmeticcompositions such that a mixture is formed or may be used as a solecomposition.

Pharmaceutical compositions may be utilized to administer the PUFAcomponent to an individual. Suitable pharmaceutical compositions maycomprise physiologically acceptable sterile aqueous or non-aqueoussolutions, dispersions, suspensions or emulsions and sterile powders forreconstitution into sterile solutions or dispersions for ingestion.Examples of suitable aqueous and non-aqueous carriers, diluents,solvents or vehicles include water, ethanol, polyols (propyleneglycol,polyethyleneglycol, glycerol, and the like), suitable mixtures thereof,vegetable oils (such as olive oil) and injectable organic esters such asethyl oleate. Proper fluidity can be maintained, for example, by themaintenance of the required particle size in the case of dispersions andby the use of surfactants. It may also be desirable to include isotonicagents, for example sugars, sodium chloride and the like. Besides suchinert diluents, the composition can also include adjuvants, such aswetting agents, emulsifying and suspending agents, sweetening, flavoringand perfuming agents.

Suspensions, in addition to the active compounds, may contain suspendingagents, as for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth or mixtures of thesesubstances, and the like.

Solid dosage forms such as tablets and capsules can be prepared usingtechniques well known in the art. For example, PUFAs of the inventioncan be tableted with conventional tablet bases such as lactose, sucrose,and cornstarch in combination with binders such as acacia, cornstarch orgelatin, disintegrating agents such as potato starch or alginic acid anda lubricant such as stearic acid or magnesium stearate. Capsules can beprepared by incorporating these excipients into a gelatin capsule alongwith the antioxidants and the PUFA component. The amount of theantioxidants and PUFA component that should be incorporated into thepharmaceutical formulation should fit within the guidelines discussedabove.

As used in this application, the term “treat” refers to eitherpreventing, or reducing the incidence of, the undesired occurrence. Forexample, to treat immune suppression refers to either preventing theoccurrence of this suppression or reducing the amount of suchsuppression. The terms “patient” and “individual” are being usedinterchangeably and both refer to an animal. The term “animal” as usedin this application refers to any warm-blooded mammal including, but notlimited to, dogs, humans, monkeys, and apes. As used in the applicationthe term “about” refers to an amount varying from the stated range ornumber by a reasonable amount depending upon the context of use. Anynumerical number or range specified in the specification should beconsidered to be modified by the term about.

“Dose” and “serving” are used interchangeably and refer to the amount ofthe nutritional or pharmaceutical composition ingested by the patient ina single setting and designed to deliver effective amounts of theantioxidants and the structured triglyceride. As will be readilyapparent to those skilled in the art, a single dose or serving of theliquid nutritional powder should supply the amount of antioxidants andPUFAs discussed above. The amount of the dose or serving should be avolume that a typical adult can consume in one sitting. This amount canvary widely depending upon the age, weight, sex or medical condition ofthe patient. However as a general guideline, a single serving or dose ofa liquid nutritional produce should be considered as encompassing avolume from 100 to 600 ml, more preferably from 125 to 500 ml and mostpreferably from 125 to 300 ml.

The PUFAs of the present invention may also be added to food even whensupplementation of the diet is not required. For example, thecomposition may be added to food of any type including but not limitedto margarines, modified butters, cheeses, milk, yogurt, chocolate,candy, snacks, salad oils, cooking oils, cooking fats, meats, fish andbeverages.

Pharmaceutical Applications

For pharmaceutical use (human or veterinary), the compositions aregenerally administered orally but can be administered by any route bywhich they may be successfully absorbed, e.g., parenterally (i.e.subcutaneously, intramuscularly or intravenously), rectally or vaginallyor topically, for example, as a skin ointment or lotion. The PUFAs ofthe present invention may be administered alone or in combination with apharmaceutically acceptable carrier or excipient. Where available,gelatin capsules are the preferred form of oral administration. Dietarysupplementation as set forth above also can provide an oral route ofadministration. The unsaturated acids of the present invention may beadministered in conjugated forms, or as salts, esters, amides orprodrugs of the fatty acids. Any pharmaceutically acceptable salt isencompassed by the present invention; especially preferred are thesodium, potassium or lithium salts. Also encompassed are theN-alkylpolyhydroxamine salts, such as N-methyl glucamine, found in PCTpublication WO 96/33155. The preferred esters are the ethyl esters. Assolid salts, the PUFAs also can be administered in tablet form. Forintravenous administration, the PUFAs or derivatives thereof may beincorporated into commercial formulations such as Intralipids. Thetypical normal adult plasma fatty acid profile comprises 6.64 to 9.46%of ARA, 1.45 to 3.11% of DGLA, and 0.02 to 0.08% of GLA. These PUFAs ortheir metabolic precursors can be administered, either alone or inmixtures with other PUFAs, to achieve a normal fatty acid profile in apatient. Where desired, the individual components of formulations may beindividually provided in kit form, for single or multiple use. A typicaldosage of a particular fatty acid is from 0.1 mg to 20 g, or even 100 gdaily, and is preferably from 10 mg to 1, 2, 5 or 10 g daily asrequired, or molar equivalent amounts of derivative forms thereof.Parenteral nutrition compositions comprising from about 2 to about 30weight percent fatty acids calculated as triglycerides are encompassedby the present invention; preferred is a composition having from about 1to about 25 weight percent of the total PUFA composition as GLA (U.S.Pat. No. 5,196,198). Other vitamins, and particularly fat-solublevitamins such as vitamin A, D, E and L-carnitine can optionally beincluded. Where desired, a preservative such as a tocopherol may beadded, typically at about 0.1% by weight.

Suitable pharmaceutical compositions may comprise physiologicallyacceptable sterile aqueous or non-aqueous solutions, dispersions,suspensions or emulsions and sterile powders for reconstitution intosterile injectible solutions or dispersions. Examples of suitableaqueous and non-aqeuous carriers, diluents, solvents or vehicles includewater, ethanol, polyols (propylleneglyol, polyethylenegycol, glycerol,and the like), suitable mixtures thereof, vegetable oils (such as oliveoil) and injectable organic esters such as ethyl oleate. Proper fluiditycan be maintained, for example, by the maintenance of the requiredparticle size in the case of dispersions and by the use of surfactants.It may also be desirable to include isotonic agents, for example sugars,sodium chloride and the like. Besides such inert diluents, thecomposition can also include adjuvants, such as wetting agents,emulsifying and suspending agents, sweetening, flavoring and perfumingagents.

Suspensions in addition to the active compounds, may contain suspendingagents, as for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, or mixtures of thesesubstances and the like.

An especially preferred pharmaceutical composition containsdiacetyltartaric acid esters of mono- and diglycerides dissolved in anaqueous medium or solvent. Diacetyltartaric acid esters of mono- anddiglycerides have an HLB value of about 9-12 and are significantly morehydrophilic than existing antimicrobial lipids that have HLB values of2-4. Those existing hydrophobic lipids cannot be formulated into aqueouscompositions. As disclosed herein, those lipids can now be solubilizedinto aqueous media in combination with diacetyltartaric acid esters ofmono- and diglycerides. In accordance with this embodiment,diacetyltartaric acid esters of mono- and diglycerides (e.g.,DATEM-C12:0) is melted with other active antimicrobial lipids (e.g.,18:2 and 12:0 monoglycerides) and mixed to obtain a homogeneous mixture.Homogeneity allows for increased antimicrobial activity. The mixture canbe completely dispersed in water. This is not possible without theaddition of diacetyltartaric acid esters of mono- and diglycerides andpremixing with other monoglycerides prior to introduction into water.The aqueous composition can then be admixed under sterile conditionswith physiologically acceptable diluents, preservatives, buffers orpropellants as may be required to form a spray or inhalant.

The present invention also encompasses the treatment of numerousdisorders with fatty acids. Supplementation with PUFAs of the presentinvention can be used to treat restenosis after angioplasty. Symptoms ofinflammation, rheumatoid arthritis, and asthma and psoriasis can betreated with the PUFAs of the present invention. Evidence indicates thatPUFAs may be involved in calcium metabolism, suggesting that PUFAs ofthe present invention may be used in the treatment or prevention ofosteoporosis and of kidney or urinary tract stones.

The PUFAs of the present invention can be used in the treatment ofcancer. Malignant cells have been shown to have altered fatty acidcompositions; addition of fatty acids has been shown to slow theirgrowth and cause cell death, and to increase their susceptibility tochemotherapeutic agents. GLA has been shown to cause reexpression oncancer cells of the E-cadherin cellular adhesion molecules, loss ofwhich is associated with aggressive metastasis. Clinical testing ofintravenous administration of the water soluble lithium salt of GLA topancreatic cancer patients produced statistically significant increasesin their survival. PUFA supplementation may also be useful for treatingcachexia associated with cancer.

The PUFAs of the present invention can also be used to treat diabetes(U.S. Pat. No. 4,826,877; Horrobin et al., Am. J. Clin. Nutr. Vol. 57(Suppl.), 732S-737S). Altered fatty acid metabolism and composition hasbeen demonstrated in diabetic animals. These alterations have beensuggested to be involved in some of the long-term complicationsresulting from diabetes, including retinopathy, neuropathy, nephropathyand reproductive system damage. Primrose oil, which contains GLA, hasbeen shown to prevent and reverse diabetic nerve damage.

The PUFAs of the present invention can be used to treat eczema, reduceblood pressure and improve math scores. Essential fatty acid deficiencyhas been suggested as being involved in eczema, and studies have shownbeneficial effects on eczema from treatment with GLA. GLA has also beenshown to reduce increases in blood pressure associated with stress, andto improve performance on arithmetic tests. GLA and DGLA have been shownto inhibit platelet aggregation, cause vasodilation, lower cholesterollevels and inhibit proliferation of vessel wall smooth muscle andfibrous tissue (Brenner et al., Adv. Exp. Med. Biol. Vol. 83, p. 85-101,1976). Administration of GLA or DGLA, alone or in combination with EPA,has been shown to reduce or prevent gastro-intestinal bleeding and otherside effects caused by non-steroidal anti-inflammatory drugs (U.S. Pat.No. 4,666,701). GLA and DGLA have also been shown to prevent or treatendometriosis and premenstrual syndrome (U.S. Pat. No. 4,758,592) and totreat myalgic encephalomyelitis and chronic fatigue after viralinfections (U.S. Pat. No. 5,116,871).

Further uses of the PUFAs of this invention include use in treatment ofAIDS, multiple schlerosis, acute respiratory syndrome, hypertension andinflammatory skin disorders. The PUFAs of the inventions also can beused for formulas for general health as well as for geriatrictreatments.

Veterinary Applications

It should be noted that the above-described pharmaceutical andnutritional compositions may be utilized in connection with animals, aswell as humans, as animals experience many of the same needs andconditions as human. For example, the oil or acids of the presentinvention may be utilized in animal feed supplements.

The following examples are presented by way of illustration, not oflimitation.

EXAMPLES Example 1 Construction of a cDNA Library from Mortierellaalpina Example 2 Isolation of a Δ6-Desaturase Nucleotide Sequence fromMortierella alpina Example 3 Identification of Δ6-Desaturases Homologousto the Mortierella alpina Δ6-Desaturase Example 4 Isolation of aΔ2-Desaturase Nucleotide Sequence from Mortierella alpina Example 5Expression of M. alpina Desaturase Clones in Baker's Yeast Example 6Initial Optimization of Culture Conditions Example 7 Distribution ofPUFAs in Yeast Lipid Fractions Example 8 Further Culture Optimizationand Coexpression of Δ6 and Δ12-Desaturases Example 9 Identification ofHomologues to M. alpina Δ5 and Δ6 Desaturases Example 10 Identificationof M. alpina Δ5 and Δ6 Homologues in other PUFA-Producing OrganismsExample 11 Identification of M. alpina Δ5 and Δ6 Homologues in otherPUFA-Producing Organisms Example 12 Human Desaturase Gene SequencesExample 13 Nutritional Compositions Example 1 Construction of a cDNALibrary from Mortierella alpina

Total RNA was isolated from a 3 day old PUFA-producing culture ofMortierella alpina using the protocol of Hoge et al. (1982) ExperimentalMycology 6:225-232. The RNA was used to prepare double-stranded cDNAusing BRL's lambda-ZipLox system following the manufacturesinstructions. Several size fractions of the M. alpina cDNA were packagedseparately to yield libraries with different average-sized inserts. A“full-length” library contains approximately 3×10⁶ clones with anaverage insert size of 1.77 kb. The “sequencing-grade” library containsapproximately 6×10⁵ clones with an average insert size of 1.1 kb.

Example 2 Isolation of a Δ6-Desaturase Nucleotide Sequence fromMortierella alpina

A nucleic acid sequence from a partial cDNA clone, Ma524, encoding a Δ6fatty acid desaturase from Mortierella alpina was obtained by randomsequencing of clones from the M. alpina cDNA sequencing grade librarydescribed in Example 1. cDNA-containing plasmids were excised asfollows:

Five μl of phage were combined with 100 μl of E. coli DH10B(ZIP) grownin ECLB plus 10 μg/ml kanamycin, 0.2% maltose, and 10 mM MgSO₄ andincubated at 37 degrees for 15 minutes. 0.9 ml SOC was added and 100 μlof the bacteria immediately plated on each of 10 ECLB+50 μg Pen plates.No 45 minute recovery time was needed. The plates were incubatedovernight at 37°. Colonies were picked into ECLB+50 μg Pen media forovernight cultures to be used for making glycerol stocks and miniprepDNA. An aliquot of the culture used for the miniprep is stored as aglycerol stock. Plating on ECLB+50 μg Pen/ml resulted in more coloniesand a greater proportion of colonies containing inserts than plating on100 μg/ml Pen.

Random colonies were picked and plasmid DNA purified using Qiagenminiprep kits. DNA sequence was obtained from the 5′ end of the cDNAinsert and compared to the National Center for Biotechnology Information(NCBI) nonredundant database using the BLASTX algorithm. Ma524 wasidentified as a putative desaturase based on DNA sequence homology topreviously identified desaturases.

A full-length cDNA clone was isolated from the M. alpina full-lengthlibrary and designed pCGN5532. The cDNA is contained as a 1617 by insertin the vector pZL1 (BRL) and, beginning with the first ATG, contains anopen reading frame encoding 457 amino acids. The three conserved“histidine boxes” known to be conserved among membrane-bound deaturases(Okuley, et al. (1994) The Plant Cell 6:147-158) were found to bepresent at amino acid positions 172-176, 209-213, and 395-399 (see FIG.3). As with other membrane-bound Δ6-desaturases the final HXXHHhistidine box motif was found to be QXXHH. The amino acid sequence ofMa524 was found to display significant homology to a portion of aCaenorhabditis elegans cosmid, WO6D2.4, a cytochrome b5/desaturasefusion protein from sunflower, and the Synechocystis and SpirulinaΔ6-desaturases. In addition, Ma524 was shown to have homology to theborage Δ6-desaturase amino sequence (PCT publication W) 96/21022). Ma524thus appears to encode a Δ6-desaturase that is related to the borage andalgal Δ6-desaturases. The peptide sequences are shown as SEQ ID NO:5-SEQID NO: 11.

The amino terminus of the encoded protein was found to exhibitsignificant homology to cytochrome b5 proteins. The Mortierella cDNAclone appears to represent a fusion between a cytochrome b5 and a fattyacid desaturase. Since cytochrome b5 is believed to function as theelectron donor for membrane-bound desaturase enzymes, it is possiblethat the N-terminal cytochrome b5 domain of this desaturase protein isinvolved in its function. This may be advantageous when expressing thedesaturase in heterologous systems for PUFA production. However, itshould be noted that, although the amino acid sequences of Ma524 and theborage Δ6 were found to contain regions of homology, the basecompositions of the cDNAs were shown to be significantly different. Forexample, the borage cDNA was shown to have an overall base compositionof 60% A/T, with some regions exceeding 70%, while Ma524 was shown tohave an average of 44% A/T base composition, with no regions exceeding60%. This may have implications for expressing the cDNAs inmicroorganisms or animals which favor different base compositions. It isknown that poor expression of recombinant genes can occur when the hostprefers a base composition different from that of the introduced gene.Mechanisms for such poor expression include decreased stability, crypticsplice sites, and/or translatability of the mRNA and the like.

Example 3 Identification of Δ6-Desaturases Homologous to the Mortierellaalpina Δ6-Desaturase

Nucleic acid sequences that encode putative Δ6-desaturases wereidentified through a BLASTX search of the Expressed Sequence Tag (“EST”)databases through NCBI using the Ma524 amino acid sequence. Severalsequences showed significant homology. In particular, the deduced aminoacid sequence of two Arabidopsis thaliana sequences, (accession numbersF13728 and T42806) showed homology to two different regions of thededuced amino acid sequence of Ma524. The following PCR primers weredesigned: ATTS4723-FOR (complementary to F13728) SEQ ID NO:13 5′CUACUACUACUAGGAGTCCTCTACGGTGTTTTG and T42806-REV (complementary toT42806) SEQ ID NO: 14 5′CAUCAUCAUCAUATGATGCTCAAGCTGAAACTG. Five μg oftotal RNA isolated from developing siliques of Arabidopsis thaliana wasreverse transcribed using BRL Superscript RTase and the primer TSyn(5′-CCAAGCTTCTGCAGGAGCTCTTTTTTTTTTTTTTT-3′) and is shown as SEQ ID NO:12. PCR was carried out in a 50 ul volume containing: template derivedfrom 25 ng total RNA, 2 pM each primer, 200 μM each deoxyribonucleotidetriphosphate, 60 mM Tris-Cl, pH 8.5, 15 mM (NH₄)₂SO₄, 2 mM MgCl₂, 0.2 UTaq Polymerase. Thermocycler conditions were as follows: 94 degrees for30 sec., 50 degrees for 30 sec., 72 degrees for 30 sec. PCR wascontinued for 35 cycles followed by an additional extension at 72degrees for 7 minutes. PCR resulted in a fragment of approximately.about.750 base pairs which was subcloned, named 12-5, and sequenced.Each end of this fragment was formed to correspond to the ArabidopsisESTs from which the PCR primers were designed. The putative amino acidsequence of 12-5 was compared to that of Ma524, and ESTs from human(W28140), mouse (W53753), and C. elegans (R05219) (see FIG. 4). Homologypatterns with the Mortierella Δ6-desaturase indicate that thesesequences represent putative desaturase polypeptides. Based on thisexperiment approach, it is likely that the full-length genes can becloned using probes based on the EST sequences. Following the cloning,the genes can then be placed into expression vectors, expressed in hostcells, and their specific Δ6- or other desaturase activity can bedetermined as described below.

Example 4 Isolation of a Δ12-Desaturase Nucleotide Sequence fromMortierella alpina

Based on the fatty acids it accumulates, it seemed probable thatMortierella alpina has an ω6 type desaturase. The ω6-desaturase isresponsible for the production of linoleic acid (18:2) from oleic acid(18:1). Linoleic acid (18:2) is a substrate for a Δ6-desaturase. Thisexperiment was designed to determine if Mortierella alpina has aΔ12-desaturase polypeptide, and if so, to identify the correspondingnucleotide sequence.

A random colony from the M. alpina sequencing grade library, Ma648, wassequenced and identified as a putative desaturase based on DNA sequencehomology to previously identified desaturases, as described for Ma524(see Example 2). The nucleotide sequence is shown in SEQ ID NO:13. Thepeptide sequence is shown in SEQ ID NO:4. The deduced amino acidsequence from the 5′ end of the Ma648 cDNA displays significant homologyto soybean microsomal ω6 (Δ12) desaturase (accession #L43921) as well ascastor bean oleate 12-hydroxylase (accession #U22378). In addition,homology was observed when compared to a variety of other ω6 (Δ12) andω3 (Δ15) fatty acid desaturase sequences.

Example 5 Expression of M. alpina Desaturase Clones in Baker's YeastYeast Transformation

Lithium acetate transformation of yeast was performed according tostandard protocols (Methods in Enzymology, Vol. 194, p. 186-187, 1991).Briefly, yeast were grown in YPD at 30° C. Cells were spun down,resuspended in TE, spun down again, resuspended in TE containing 100 mMlithium acetate, spun down again, and resuspended in TE/lithium acetate.The resuspended yeast were incubated at 30° C. for 60 minutes withshaking. Carrier DNA was added, and the yeast were aliquoted into tubes.Transforming DNA was added, and the tubes were incubated for 30 min. at30° C. PEG solution (35% (w/v) PEG 4000, 100 mM lithium acetate, TEpH7.5) was added followed by a 50 min. incubation at 30° C. A 5 min.heat shock at 42° C. was performed, the cells were pelleted, washed withTE, pelleted again and resuspended in TE. The resuspended cells werethen plated on selective media.

Desaturase Expression in Transformed Yeast

cDNA clones from Mortierella alpina were screened for desaturaseactivity in baker's yeast. A canola Δ15-desaturase (obtained by PCRusing 1^(st) strand cDNA from Brassica napus cultivar 212/86 seeds usingprimers based on the published sequence (Arondel et al. Science258:1353-1355)) was used as a positive control. The Δ15-desaturase geneand the gene from cDNA clones Ma524 and Ma648 were put in the expressionvector pYES2 (Invitrogen), resulting in plasmids pCGR-2, pCGR-5 andpCGR-7, respectively. These plasmids were transfected into S. cerevisiaeyeast strain 334 and expressed after induction with galactose and in thepresence of substrates that allowed detection of specific desaturaseactivity. The control strain was S. cerevisiae strain 334 containing theunaltered pYES2 vector. The substrates used, the products produced andthe indicated desaturase activity were: DGLA (conversion to ARA wouldindicate Δ5-desaturase activity), linoleic acid (conversion to GLA wouldindicate Δ6-desaturase activity; conversion to ALA would indicateΔ15-desaturase activity), oleic acid (an endogenous substrate made by S.cerevisiae, conversion to linoleic acid would indicate Δ12-desaturaseactivity, which S. cerevisiae lacks), or ARA (conversion to EPA wouldindicate Δ17-desaturase activity).

Cultures were grown for 48-52 hours at 15° C. in the presence of aparticular substrate. Lipid fractions were extracted for analysis asfollows: Cells were pelleted by centrifugation, washed once with sterileddH₂0, and repelleted. Pellets were vortexed with methanol; chloroformwas added along with tritridecanoin (as an internal standard). Themixtures were incubated for at least one hour at room temperature or at4° C. overnight. The chloroform layer was extracted and filtered througha Whatman filter with one gram of anhydrous sodium sulfate to removeparticulates and residual water. The organic solvents were evaporated at40° C. under a stream of nitrogen. The extracted lipids were thenderivatized to fatty acid methyl esters (FAME) for gas chromatographyanalysis (GC) by adding 2 ml of 0.5 N potassium hydroxide in methanol toa closed tube. The samples were heated to 95° C. to 100° C. for 30minutes and cooled to room temperature. Approximately 2 ml of 14% borontrifluoride in methanol was added and the heating repeated. After theextracted lipid mixture cooled, 2 ml of water and 1 ml of hexane wereadded to extract the FAME for analysis by GC. The percent conversion wascalculated by dividing the product produced by the sum of (the productproduced and the substrate added) and then multiplying by 100. Tocalculate the oleic acid percent conversion, as no substrate was added,the total linoleic acid produced was divided by the sum of oleic acidand linoleic acid produced, then multiplying by 100. The desaturaseactivity results are provided in Table 1 below. TABLE 1 M. alpinaDesaturase Expression in Baker's Yeast % CONVERSION CLONE ENZYMEACTIVITY OF SUBSTRATE pCGR-2 Δ6   0 (18:2 to 18:3w6) (canola Δ15 Δ1516.3 (18:2 to 18:3w3)  desaturase) Δ5  2.0 (20:3 to 20:4w6) Δ17 2.8(20:4 to 20:5w3) Δ12 1.8 (18:1 to 18:2w6) pCGR-5 (M. Δ6  6.0 alpinaMa524 Δ15 0 Δ5  2.1 Δ17 0 Δ12 3.3 pCGR-7 (M. Δ6  0 alpina Ma648 Δ15 3.8Δ5  2.2 Δ17 0 Δ12 63.4

The Δ15-desaturase control clone exhibited 16.3% conversion of thesubstrate. The pCGR-5 clone expressing the Ma524 cDNA showed 6%conversion of the substrate to GLA, indicating that the gene encodes aΔ6-desaturase. The pCGR-7 clone expressing the Ma648 cDNA converted63.4% conversion of the substrate to LA, indicating that the geneencodes a Δ12-desaturase. The background (non-specific conversion ofsubstrate) was between 0-3% in these cases. We also found substrateinhibition of the activity by using different concentrations of thesubstrate. When substrate was added to 100 μM, the percent conversion toproduct dropped compared to when substrate was added to 25 μM (seebelow). Additionally, by varying the substrate concentration between 5μM and 200 μM, conversion ratios were found to range between about 5% toabout 75% greater. These data show that desaturases with differentsubstrate specificities can be expressed in a heterologous system andused to produce poly-unsaturated long chain fatty acids.

Table 2 represents fatty acids of interest as a percent of the totallipid extracted from the yeast host S. cerevisiae 334 with the indicatedplasmid. No glucose was present in the growth media. Affinity gaschromatography was used to separate the respective lipids. GC/MS wasemployed to verify the identity of the product(s). The expected productfor the B. napus Δ15-desaturase, α-linolenic acid, was detected when itssubstrate, linoleic acid, was added exogenously to the induced yeastculture. This finding demonstrates that yeast expression of a desaturasegene can produce functional enzyme and detectable amounts of productunder the current growth conditions. Both exogenously added substrateswere taken up by yeast, although slightly less of the longer chain PUFA,dihomo-γ-linolenic acid (20:3), was incorporated into yeast thanlinoleic acid (18:2) when either was added in free form to the inducedyeast cultures. γ-linolenic acid was detected when linoleic acid waspresent during induction and expression of S. cerevisiae 334 (pCGR-5).The presence of this PUFA demonstrates Δ6-desaturase activity frompCGR-5 (MΔ524). Linoleic acid, identified in the extracted lipids fromexpression of S. cerevisiae 334 (pCGR-7), classifies the cDNA MΔ648 fromM. alpina as the Δ12-desaturase. TABLE 2 Fatty Acid as a Percentage ofTotal Lipid Extracted from Yeast Plasmid in Yeast 18:2 α-18:3 γ-l8:320:3 20:4 18:1* 18:2 (enzyme) Incorporated Produced ProducedIncorporated Produced Present Produced pYES2 66.9 0 0 58.4 0 4 0(control) pCGR-2 60.1 5.7 0 50.4 0 0.7 0 (Δ15) pCGR-5 62.4 0 4.0 49.9 02.4 0 (Δ6) pCGR-7 65.6 0 0 45.7 0 7.1 12.2 (Δ12)100 μM substrate added*18:1 is an endogenous fatty acid in yeastKey To Tables18:1 = oleic acid18:2 = linoleic acidα-18:3 = α-linolenic acidγ-18:3 = γ-linolenic acid18:4 = stearidonic acid20:3 = dihomo-γ-linolenic acid20:4 = arachidonic acid

Example 6 Optimization of Culture Conditions

Table 3A shows the effect of exogenous free fatty acid substrateconcentration on yeast uptake and conversion to fatty acid product as apercentage of the total yeast lipid extracted. In all instances, lowamounts of exogenous substrate (1-10 μM) resulted in low fatty acidsubstrate uptake and product formation. Between 25 and 50 μMconcentration of free fatty acid in the growth and induction media gavethe highest percentage of fatty acid product formed, while the 100 μMconcentration and subsequent high uptake into yeast appeared to decreaseor inhibit the desaturase activity. The amount of fatty acid substratefor yeast expressing Δ12-desaturase was similar under the same growthconditions, since the substrate, oleic acid, is an endogenous yeastfatty acid. The use of α-linolenic acid as an additional substrate forpCGR-5 (Δ6) produced the expected product, stearidonic acid (Table 3A).The feedback inhibition of high fatty acid substrate concentration waswell illustrated when the percent conversion rates of the respectivefatty acid substrates to their respective products were compared inTable 3B. In all cases, 100 μM substrate concentration in the growthmedia decreased the percent conversion to product. The uptake ofα-linolenic was comparable to other PUFAs added in free form, while theΔ6-desaturase percent conversion, 3.8-17.5%, to the product stearidonicacid was the lowest of all the substrates examined (Table 3B). Theeffect of media, such as YPD (rich media) versus minimal media withglucose on the conversion rate of Δ12-desaturase was dramatic. Not onlydid the conversion rate for oleic to linoleic acid drop, (Table 3B) butthe percent of linoleic acid formed also decreased by 11% when richmedia was used for growth and induction of yeast desaturase Δ12expression (Table 3A). The effect of media composition was also evidentwhen glucose was present in the growth media for Δ6-desaturase, sincethe percent of substrate uptake was decreased at 25 μM (Table 3A).However, the conversion rate remained the same and percent productformed decreased for Δ6-desaturase for in the presence of glucose. TABLE3A Effect of Added Substrate on the Percentage of Incorporated Substrateand Product Formed in Yeast Extracts Plasmid in Yeast pCGR-2 PcGR-5pCGR-5 pCGR-7 (Δ15) (Δ6) (Δ6) (Δ12) Substrate/ 18:2/α-18:3 18:2/γ-18:3α-18:3/18:4 18:1*/18:2 product 1 μM sub. ND 0.9/0.7 ND ND 10 μM sub. ND4.2/2.4 10.4/2.2 ND 25 μM sub. ND 11/3.7 18.2/2.7 ND 25 μM ⋄ sub.36.6/7.2 ⋄ 25.1/10.3 ⋄ ND 6.6/15.8 ⋄ 50 μM sub. 53.1/6.5 ⋄ ND 36.2/310.8/13⁺ 100 μM sub. 60.1/5.7 ⋄ 62.4/4 ⋄ 47.7/1.9 10/24.8

TABLE 3B Effect of Substrate Concentration in Media on the PercentConversion of Fatty Acid Substrate to Product in Yeast Extracts Plasmidin Yeast pCGR-2 pCGR-5 pCGR-5 pCGR-7 (Δ15) (Δ6) (Δ6) (Δ12)substrate→product 18:2→α-18:3 18:2→γ18:3 α-18:3→18:4 18:1*→18:2 1 μMsub. ND 43.8 ND ND 10 μM sub. ND 36.4 17.5 ND 25 μM sub. ND 25.2 12.9 ND25 μM ⋄ sub. 16.4 ⋄  29.1 ⋄ ND 70.5 ⋄ 50 μM sub. 10.9 ⋄ ND 7.7 54.6⁺ 100 μM sub.  8.7 ⋄   6 ⋄ 3.8 71.3 ⋄no glucose in media⁺Yeast peptone broth (YPD)*18:1 is an endogenous yeast lipidsub. is substrate concentrationND (not done)

Table 4 shows the amount of fatty acid produced by a recombinantdesaturase from induced yeast cultures when different amounts of freefatty acid substrate were used. Fatty acid weight was determined sincethe total amount of lipid varied dramatically when the growth conditionswere changed, such as the presence of glucose in the yeast growth andinduction media. To better determine the conditions when the recombinantdesaturase would produce the most PUFA product, the quantity ofindividual fatty acids were examined. The absence of glucosedramatically reduced by three fold the amount of linoleic acid producedby recombinant Δ12-desaturase. For the Δ12-desaturase the amount oftotal yeast lipid was decreased by almost half in the absence ofglucose. Conversely, the presence of glucose in the yeast growth mediafor Δ6-desaturase drops the γ-linolenic acid produced by almost half,while the total amount of yeast lipid produced was not changed by thepresence/absence of glucose. This points to a possible role for glucoseas a modulator of Δ6-desaturase activity. TABLE 4 Fatty Acid Produced inμg from Yeast Extracts Plasmid in Yeast (enzyme) pCGR-5 pCGR-5 pCGR-7(Δ6) (Δ6) (Δ12) product Y-18:3 18:4 18:2*  1 μM sub. 1.9 ND ND 10 μMsub. 5.3 4.4 ND 25 μM sub. 10.3 8.7 115.7 25 μM sub. 29.6 ND 39 ⋄⋄no glucose in mediasub. is substrate concentrationND (not done)*18:1, the substrate, is an endogenous yeast lipid

Example 7 Distribution of PUFAs in Yeast Lipid Fractions

Table 5 illustrates the uptake of free fatty acids and their newproducts formed in yeast lipids as distributed in the major lipidfractions. A total lipid extract was prepared as described above. Thelipid extract was separated on TLC plates, and the fractions wereidentified by comparison to standards. The bands were collected byscraping, and internal standards were added. The fractions were thensaponified and methylated as above, and subjected to gas chromatography.The gas chromatograph calculated the amount of fatty acid by comparisonto a standard. The phospholipid fraction contained the highest amount ofsubstrate and product PUFAs for Δ6-desaturase activity. It would appearthat the substrates are accessible in the phospholipid form to thedesaturases. TABLE 5 Fatty Acid Distribution in Various Yeast LipidFractions in μg Free Choles- Fatty acid Phopho- Fatty terol fractionlipid Diglyceride Acid Triglyceride Ester SC (pCGR-5) 166.6 6.2 15 18.215.6 substrate 18:2 SC (pCGR-5) 61.7 1.6 4.2 5.9 1.2 product γ-18:3SC = S. cerevisiae (plasmid)

Example 8 Further Culture Optimization and Coexpression of Δ6 andΔ12-Desaturases

This experiment was designed to evaluate the growth and inductionconditions for optimal activities of desaturases in Saccharomycescerevisiae. A Saccharomyces cerevisiae strain (SC334) capable ofproducing γ-linolenic acid (GLA) was developed, to assess thefeasibility of production of PUFA in yeast. The genes for Δ6 andΔ12-desaturases from M. alpina were coexpressed in SC334. Expression ofΔ12-desaturase converted oleic acid (present in yeast) to linoleic acid.The linoleic acid was used as a substrate by the Δ6-desaturase toproduce GLA. The quantity of GLA produced ranged between 5-8% of thetotal fatty acids produced in SC334 cultures and the conversion rate oflinoleic acid to γ-linolenic acid ranged between 30% to 50%. Theinduction temperature was optimized, and the effect of changing hoststrain and upstream promoter sequences on expression of Δ6 and Δ12 (MA524 and MA 648 respectively) desaturase genes was also determined.

Plasmid Construction

The cloning of pCGR5 as well as pCGR7 has been discussed above. Toconstruct pCGR9a and pCGR9b, the Δ6 and Δ12-desaturase genes wereamplified using the following sets of primers. The primers pRDS1 and 3had Xho1 site and primers pRDS2 and 4 had Xba1 site (indicated in bold).These primer sequences are presented as SEQ ID NO:15-18. I.Δ6-Desaturase Amplification Primers a. pRDS1 TAC CAA CTC GAG AAA ATG GCTGCT GCT CCC AGT GTG AGG b. pRDS2 AAC TGA TCT AGA TTA CTG CGC CTT ACC CATCTT GGA GGC II. Δ12-Desaturase Amplification Primers a. pRDS3 TAC CAACTC GAG AAA ATG GCA CCT CCC AAC ACT ATC GAT b. pRDS4 AAC TGA TCT AGA TTACTT CTT GAA AAA GAC CAC GTC TCC

The pCGR5 and pCGR7 constructs were used as template DNA foramplification of Δ6 and Δ12-desaturase genes, respectively. Theamplified products were digested with Xba1 and XhoI to create “stickyends”. The PCR amplified Δ6-desaturase with Xho1-Xba1 ends as clonedinto pCGR7, which was also cut with Xho-1-Xba1. This procedure placedthe Δ6-desaturase behind the Δ12-desaturase, under the control of aninducible promoter GAL1. This construct was designated pCGR9a.Similarly, to construct pCGR9b, the Δ12-desaturase with XhoI-XbaI endswas cloned in the XhoI-XbaI sites of pCGR5. In pCGR9b the Δ12-desaturasewas behind the Δ6-desaturase gene, away from the GAL promoter.

To construct pCGR10, the vector pRS425, which contains the constitutiveGlyceraldehyde 3-Phosphate Dehydrogenase (GPD) promoter, was digestedwith BamH1 and pCGR5 was digested with BamH1H-Xho1 to release theΔ6-desaturase gene. This Δ6-desaturase fragment and BamH1 cut pRS425were filled using Klenow Polymerase to create blunt ends and ligated,resulting in pCGR10a and pCGR10b containing the Δ6-desaturase gene inthe sense and antisense orientation, respectively. To construct pCGR11and pCGR12, the Δ6 and Δ12-desaturase genes were isolated from pCGR5 andpCGR7, respectively, using an EcoR1-XhoI double digest. The EcoR1-Xho1fragments of Δ6 and Δ12-desaturases were cloned into the pYX242 vectordigested with EcoR1-Xho1. The pYX242 vector has the promoter of TP1 (ayeast housekeeping gene), which allows constitutive expression.

Yeast Transformation and Expression

Different combinations of pCGR5, pCGR7, pCGR9a, pCGR9b, pCGR10a, pCGR11and pCGR12 were introduced into various host strains of Saccharomycescerevisiae. Transformation was done using PEG/LiAc protocol (Methods inEnzymology Vol. 194 (1991): 186-187). Transformants were selected byplating on synthetic media lacking the appropriate amino acid. ThepCGR5, pCGR7, pCGR9a and pCGR9b can be selected on media lacking uracil.The pCGR10, pCGR11 and pCGR12 constructs can be selected on medialacking leucine. Growth of cultures and fatty acid analysis wasperformed as in Example 5 above.

Production of GLA

Production of GLA requires the expression of two enzymes (the Δ6 andΔ12-desaturases), which are absent in yeast. To express these enzymes atoptimum levels the following constructs or combinations of constructs,were introduced into various host strains:

1) pCGR9a/SC334

2) pCGR9b/SC334

3) pCGR10a and pCGR7/SC334

4) pCGR11 and pCGR7/SC334

5) pCGR12 and pCGR5/SC334

6) pCGR10a and pCGR7/DBY746

7) pCGR10a and pCGR7/DBY746

The pCGR9a construct has both the Δ6 and Δ12-desaturase genes under thecontrol of an inducible GAL promoter. The SC334 host cells transformedwith this construct did not show any GLA accumulation in total fattyacids (FIGS. 6A and B, lane 1). However, when the Δ6 and Δ12-desaturasegenes were individually controlled by the GAL promoter, the controlconstructs were able to express Δ6- and Δ12-desaturase, as evidenced bythe conversion of their respective substrates to products. TheΔ12-desaturase gene in pCGR9a was expressed as evidenced by theconversion of 18:1ω9 to 18:2ω6 in pCGR9a/SC334, while the Δ6-desaturasegene was not expressed/active, because the 18:2ω6 was not beingconverted to 18:3ω6 (FIGS. 6A and B, lane 1).

The pCGR9b construct also had both the Δ6 and Δ12-desaturase genes underthe control of the GAL promoter but in an inverse order compared topCGR9a. In this case, very little GLA (<1%) was seen in pCGR9b/SC334cultures. The expression of Δ12-desaturase was also very low, asevidenced by the low percentage of 18:2ω6 in the total fatty acids(FIGS. 6A and B, lane 1).

To test if expressing both enzymes under the control of independentpromoters would increase GLA production, the Δ6-desaturase gene wascloned into the pRS425 vector. The construct of pCGR10a has theΔ6-desaturase in the correct orientation, under control of constitutiveGPD promoter. The pCGR10b has the Δ6-desaturase gene in the inverseorientation, and serves as the negative control. The pCGR10a/SC334 cellsproduced significantly higher levels of GLA (5% of the total fattyacids, FIG. 6, lane 3), compared to pCGR9a. Both the Δ6 andΔ12-desaturase genes were expressed at high level because the conversionof 18:1ω9→8:2ω6 was 65%, while the conversion of 18:2ω6→18:3ω6(Δ6-desaturase) was 30% (FIG. 6, lane 3). As expected, the negativecontrol pCGR10b/SC334 did not show any GLA.

To further optimize GLA production, the Δ6 and Δ12 genes were introducedinto the pYX242 vector, creating pCGR11 and pCGR12 respectively. ThepYX242 vector allows for constitutive expression by the TP1 promoter(Alber, T. and Kawasaki, G. (1982). J. Mol. & Appl. Genetics 1: 419).The introduction of pCGR11 and pCGR7 in SC334 resulted in approximately8% of GLA in total fatty acids of SC334. The rate of conversion of18:1ω9→18:2ω6 and 18:2ω6→18:3ω6 was approximately 50% and 44%respectively (FIGS. 6A and B, lane 4). The presence of pCGR12 and pCGR5in SC334 resulted in 6.6% GLA in total fatty acids with a conversionrate of approximately 50% for both 18:1ω9 to 18:2ω6 and 18:2ω6 to18:3ω6, respectively (FIGS. 6A and B, lane 5). Thus although thequantity of GLA in total fatty acids was higher in the pCGR11/pCGR7combination of constructs, the conversion rates of substrate to productwere better for the pCGR12/pCGR5 combination.

To determine if changing host strain would increase GLA production,pCGR10a and pCGR7 were introduced into the host strain BJ1995 and DBY746(obtained from the Yeast Genetic Stock Centre, 1021 Donner Laboratory,Berkeley, Calif. 94720. The genotype of strain DBY746 is Matα, his3-Δ1,leu2-3, leu2-112, ura3-32, trp1-289, gal). The results are shown in FIG.7. Changing host strain to BJ1995 did not improve the GLA production,because the quantity of GLA was only 1.31% of total fatty acids and theconversion rate of 18:1ω9→18:2ω6 was approximately 17% in BJ1995. No GLAwas observed in DBY746 and the conversion of 18:1ω9→18:2ω6 was very low(<1% in control) suggesting that a cofactor required for the expressionof Δ12-desaturase might be missing in DB746 (FIG. 7, lane 2).

To determine the effect of temperature on GLA production, SC334 culturescontaining pCGR10a and pCGR7 were grown at 15° C. and 30° C. Higherlevels of GLA were found in cultures grown and induced at 15° C. thanthose in cultures grown at 30° C. (4.23% vs. 1.68%). This was due to alower conversion rate of 18:2ω6→18:3ω6 at 30° C. (11.6% vs. 29% in 15°C.) cultures, despite a higher conversion of 18:1ω9→18:2ω6 (65% vs. 60%at 30° C. (FIG. 8). These results suggest that Δ12- and Δ6-desaturasesmay have different optimal expression temperatures.

Of the various parameters examined in this study, temperature of growth,yeast host strain and media components had the most significant impacton the expression of desaturase, while timing of substrate addition andconcentration of inducer did not significantly affect desaturaseexpression.

These data show that two DNAs encoding desaturases that can convert LAto GLA or oleic acid to LA can be isolated from Mortierella alpina andcan be expressed, either individually or in combination, in aheterologous system and used to produce poly-unsaturated long chainfatty acids. Exemplified is the production of GLA from oleic acid byexpression of Δ12- and Δ6-desaturases in yeast.

Example 9 Identification of Homologues to M. alpina Δ5 and Δ6Desaturases

A nucleic acid sequence that encodes a putative Δ5 desaturase wasidentified through a TBLASTN search of the expressed sequence tagdatabases through NCBI using amino acids 100-446 of Ma29 as a query. Thetruncated portion of the Ma29 sequence was used to avoid picking uphomologies based on the cytochrome b5 portion at the N-terminus of thedesaturase. The deduced amino acid sequence of an est from Dictyosteliumdiscoideum (accession #C25549) shows very significant homology to Ma29and lesser, but still significant homology to Ma524. The DNA sequence ispresented as SEQ ID NO: 19. The amino acid sequence is presented as SEQID NO:20.

Example 10 Identification of M. alpina Δ5 and Δ6 Homologues in OtherPUFA-Producing Organisms

To look for desaturases involved in PUFA production, a cDNA library wasconstructed from total RNA isolated from Phaeodactylum tricornutum. Aplasmid-based cDNA library was constructed in pSPORT1 (GIBCO-BRL)following manufacturer's instructions using a commercially available kit(GIBCO-BRL). Random cDNA clones were sequenced and nucleic acidsequences that encode putative Δ5 or Δ6 desaturases were identifiedthrough BLAST search of the databases and comparison to Ma29 and Ma524sequences.

One clone was identified from the Phaeodactylum library with homology toMa29 and Ma524; it is called 144-011-B12. The DNA sequence is presentedas SEQ ID NO:21. The amino acid sequence is presented as SEQ ID NO:22.

Example 11 Identification of M. alpina Δ5 and Δ6 Homologues in OtherPUFA-Producing Organisms

To look for desaturases involved in PUFA production, a cDNA library wasconstructed from total RNA isolated from Schizochytrium species. Aplasmid-based cDNA library was constructed in pSPORT1 (GIBCO-BRL)following manufacturer's instructions using a commercially available kit(GIBCO-BRL). Random cDNA clones were sequenced and nucleic acidsequences that encode putative Δ5 or Δ6 desaturases were identifiedthrough BLAST search of the databases and comparison to Ma29 and Ma524sequences.

One clone was identified from the Schizochytrium library with homologyto Ma29 and Ma524; it is called 81-23-C7. This clone contains a ˜1 kbinsert. Partial sequence was obtained from each end of the clone usingthe universal forward and reverse sequencing primers. The DNA sequencefrom the forward primer is presented as SEQ ID NO:23. The peptidesequence is presented as SEQ ID NO:24. The DNA sequence from the reverseprimer is presented as SEQ ID NO:25. The amino acid sequence from thereverse primer is presented as SEQ ID NO:26.

Example 12 Human Desaturase Gene Sequences

Human desaturase gene sequences potentially involved in long chainpolyunsaturated fatty acid biosynthesis were isolated based on homologybetween the human cDNA sequences and Mortierella alpina desaturase genesequences. The three conserved “histidine boxes” known to be conservedamong membrane-bound desaturases were found. As with some othermembrane-bound desaturases the final HXXHH histidine box motif was foundto be QXXHH. The amino acid sequence of the putative human desaturasesexhibited homology to M. alpina Δ5, Δ6, Δ9, and Δ12 desaturases.

The M. alpina Δ5 desaturase and Δ6 desaturase cDNA sequences were usedto search the LifeSeq database of Incyte Pharmaceuticals, Inc., PaloAlto, Calif. 94304. The Δ5 desaturase sequence was divided intofragments; 1) amino acid no. 1-150, 2) amino acid no. 151-300, and 3)amino acid no. 301-446. The Δ6 desaturase sequence was divided intothree fragments; 1) amino acid no. 1-150, 2) amino acid no. 151-300, and3) amino acid no. 301-457. These polypeptide fragments were searchedagainst the database using the “tblastn” algorithm. This algorithmcompares a protein query sequence against a nucleotide sequence databasedynamically translated in all six reading frames (both strands).

The polypeptide fragments 2 and 3 of M. alpina Δ5 and Δ6 have homologieswith the CloneID sequences as outlined in Table 6. The CloneIDrepresents an individual sequence from the Incyte LifeSeq database.After the “tblastn” results have been reviewed, Clone Information wassearched with the default settings of Stringency of >=50, andProductscore<=100 for different CloneID numbers. The Clone InformationResults displayed the information including the ClusterID, CloneID,Library, HitID, Hit Description. When selected, the ClusterID numberdisplayed the clone information of all the clones that belong in thatClusterID. The Assemble command assembles all of the CloneID whichcomprise the ClusterID. The following default settings were used for GCG(Genetics Computer Group, University of Wisconsin Biotechnology Center,Madison, Wis. 53705) Assembly: Word Size: 7 Minimum Overlap: 14Stringency: 0.8 Minimum Identity: 14 Maximum Gap: 10 Gap Weight: 8Length Weight: 2

GCG Assembly Results displayed the contigs generated on the basis ofsequence information within the CloneID. A contig is an alignment of DNAsequences based on areas of homology among these sequences. A newsequence (consensus sequence) was generated based on the aligned DNAsequences within a contig. The contig containing the CloneID wasidentified, and the ambiguous sites of the consensus sequence was editedbased on the alignment of the CloneIDs (see SEQ ID NO:27-SEQ ID NO:32)to generate the best possible sequence. The procedure was repeated forall six CloneID listed in Table 6. This produced five unique contigs.The edited consensus sequences of the 5 contigs were imported into theSequencher software program (Gene Codes Corporation, Ann Arbor, Mich. 48105). These consensus sequences were assembled. The contig 2511785overlaps with contig 3506132, and this new contig was called 2535 (SEQID NO:33). The contigs from the Sequencher program were copied into theSequence Analysis software package of GCG.

Each contig was translated in all six reading frames into proteinsequences. The M. alpina Δ5 (MΔ29) and Δ6 (MΔ524) sequences werecompared with each of the translated contigs using the FastA search (aPearson and Lipman search for similarity between a query sequence and agroup of sequences of the same type (nucleic acid or protein)). Homologyamong these sequences suggest the open reading frames of each contig.The homology among the M. alpina Δ5 and Δ6 to contigs 2535 and 3854933were utilized to create the final contig called 253538a. FIG. 13 is theFastA match of the final contig 253538a and MΔ29, and FIG. 14 is theFastA match of the final contig 253538a and MΔ524. The DNA sequences forthe various contigs are presented in SEQ ID NO:27-SEQ ID NO:33 Thevarious peptide sequences are shown in SEQ ID NO:34-SEQ ID NO: 40.

Although the open reading frame was generated by merging the twocontigs, the contig 2535 shows that there is a unique sequence in thebeginning of this contig which does not match with the contig 3854933.Therefore, it is possible that these contigs were generated fromindependent desaturase like human genes.

The contig 253538a contains an open reading frame encoding 432 aminoacids. It starts with Gln (CAG) and ends with the stop codon (TGA). Thecontig 253538a aligns with both M. alpina Δ5 and Δ6 sequences,suggesting that it could be either of the desaturases, as well as otherknown desaturases which share homology with each other. The individualcontigs listed in Table 18, as well as the intermediate contig 2535 andthe final contig 253538a can be utilized to isolate the complete genesfor human desaturases.

Uses of the Human Desaturases

These human sequences can be express in yeast and plants utilizing theprocedures described in the preceding examples. For expression inmammalian cells transgenic animals, these genes may provide superiorcodon bias.

In addition, these sequences can be used to isolate related desaturasegenes from other organisms. TABLE 6 Sections of the Desaturases Clone IDfrom LifeSeq Database Keyword 151-300 Δ5 3808675 fatty acid desaturase301-446 Δ5 354535 Δ6 151-300 Δ6 3448789 Δ6 151-300 Δ6 1362863 Δ6 151-300Δ6 2394760 Δ6 301-457 Δ6 3350263 Δ6

Example 13 Infant Formulations

A. Isomil® Soy Formula with Iron.

Usage: As a beverage for infants, children and adults with an allergy orsensitivity to cow's milk. A feeding for patients with disorders forwhich lactose should be avoided: lactase deficiency, lactose intoleranceand galactosemia.

Features:

Soy protein isolate to avoid symptoms of cow's-milk-protein allergy orsensitivity

Lactose-free formulation to avoid lactose-associated diarrhea

Low osmolality (240 mOsm/kg water) to reduce risk of osmotic diarrhea.

Dual carbohydrates (corn syrup and sucrose) designed to enhancecarbohydrate absorption and reduce the risk of exceeding the absorptivecapacity of the damaged gut.

1.8 mg of Iron (as ferrous sulfate) per 100 Calories to help preventiron deficiency.

Recommended levels of vitamins and minerals.

Vegetable oils to provide recommended levels of essential fatty acids.

Milk-white color, milk-like consistency and pleasant aroma.

Ingredients: (Pareve, ^({circle around (ll)})) 85% water, 4.9% cornsyrup, 2.6% sugar (sucrose), 2.1% soy oil, 1.9% soy protein isolate,1.4% coconut oil, 0.15% calcium citrate, 0.11% calcium phosphatetribasic, potassium citrate, potassium phosphate monobasic, potassiumchloride, mono- and disglycerides, soy lecithin, carrageenan, ascorbicacid, L-methionine, magnesium chloride, potassium phosphate dibasic,sodium chloride, choline chloride, taurine, ferrous sulfate, m-inositol,alpha-tocopheryl acetate, zinc sulfate, L-carnitine, niacinamide,calcium pantothenate, cupric sulfate, vitamin A palmitate, thiaminechloride hydrochloride, riboflavin, pyridoxine hydrochloride, folicacid, manganese sulfate, potassium iodide, phylloquinone, biotin, sodiumselenite, vitamin D₃ and cyanocobalamin.

B. Isomil® DF Soy Formula for Diarrhea.

Usage: As a short-term feeding for the dietary management of diarrhea ininfants and toddlers.

Features:

First infant formula to contain added dietary fiber from soy fiberspecifically for diarrhea management.

Clinically shown to reduce the duration of loose, watery stools duringmild to severe diarrhea in infants.

Nutritionally complete to meet the nutritional needs of the infant.

Soy protein isolate with added L-methionine meets or exceeds an infant'srequirement for all essential amino acids.

Lactose-free formulation to avoid lactose-associated diarrhea.

Low osmolality (240 mOsm/kg water) to reduce the risk of osmoticdiarrhea.

Dual carbohydrates (corn syrup and sucrose) designed to enhancecarbohydrate absorption and reduce the risk of exceeding the absorptivecapacity of the damaged gut.

Meets or exceeds the vitamin and mineral levels recommended by theCommittee on Nutrition of the American Academy of Pediatrics andrequired by the Infant Formula Act.

1.8 mg of iron (as ferrous sulfate) per 100 Calories to help preventiron deficiency.

Vegetable oils to provide recommended levels of essential fatty acids.

Ingredients: (Pareve, ^({circle around (ll)})) 86% water, 4.8% cornsyrup, 2.5% sugar (sucrose), 2.1% soy oil, 2.0% soy protein isolate,1.4% coconut oil, 0.77% soy fiber, 0.12% calcium citrate, 0.11% calciumphosphate tribasic, 0.10% potassium citrate, potassium chloride,potassium phosphate monobasic, mono- and disglycerides, soy lecithin,carrageenan, magnesium chloride, ascorbic acid, L-methionine, potassiumphosphate dibasic, sodium chloride, choline chloride, taurine, ferroussulfate, m-inositol, alpha-tocopheryl acetate, zinc sulfate,L-carnitine, niacinamide, calcium pantothenate, cupric sulfate, vitaminA palmitate, thiamine chloride hydrochloride, riboflavin, pyridoxinehydrochloride, folic acid, manganese sulfate, potassium iodide,phylloquinone, biotin, sodium selenite, vitamin D₃ and cyanocobalamin.

C. Isomil® SF Sucrose-Free Soy Formula with Iron.

Usage: As a beverage for infants, children and adults with an allergy orsensitivity to cow's-milk protein or an intolerance to sucrose. Afeeding for patients with disorders for which lactose and sucrose shouldbe avoided.

Features:

Soy protein isolate to avoid symptoms of cow's-milk-protein allergy orsensitivity.

Lactose-free formulation to avoid lactose-associated diarrhea(carbohydrate source is Polycose® Glucose Polymers).

Sucrose free for the patient who cannot tolerate sucrose.

Low osmolality (180 mOsm/kg water) to reduce risk of osmotic diarrhea.

1.8 mg of iron (as ferrous sulfate) per 100 Calories to help preventiron deficiency.

Recommended levels of vitamins and minerals.

Vegetable oils to provide recommended levels of essential fatty acids.

Milk-white color, milk-like consistency and pleasant aroma.

Ingredients: (Pareve, ^({circle around (ll)})) 75% water, 11.8%hydrolized cornstarch, 4.1% soy oil, 4.1% soy protein isolate, 2.8%coconut oil, 1.0% modified cornstarch, 0.38% calcium phosphate tribasic,0.17% potassium citrate, 0.13% potassium chloride, mono- anddisglycerides, soy lecithin, magnesium chloride, abscorbic acid,L-methionine, calcium carbonate, sodium chloride, choline chloride;carrageenan, taurine, ferrous sulfate, m-inositol, alpha-tocopherylacetate, zinc sulfate, L-carnitine, niacinamide, calcium pantothenate,cupric sulfate, vitamin A palmitate, thiamine chloride hydrochloride,riboflavin, pyridoxine hydrochloride, folic acid, manganese sulfate,potassium iodide, phylloquinone, biotin, sodium selenite, vitamin D₃ andcyanocobalamin.

D. Isomil® 20 Soy Formula with Iron Ready to Feed, 20 Cal/fl oz.

Usage: When a soy feeding is desired.

Ingredients: (Pareve, ^({circle around (ll)})) 85% water, 4.9% cornsyrup, 2.6% sugar (sucrose), 2.1% soy oil, 1.9% soy protein isolate,1.4% coconut oil, 0.15% calcium citrate, 0.11% calcium phosphatetribasic, potassium citrate, potassium phosphate monobasic, potassiumchloride, mono- and disglycerides, soy lecithin, carrageenan, abscorbicacid, L-methionine, magnesium chloride, potassium phosphate dibasic,sodium chloride, choline chloride, taurine, ferrous sulfate, m-inositol,alpha-tocopheryl acetate, zinc sulfate, L-carnitine, niacinamide,calcium pantothenate, cupric sulfate, vitamin A palmitate, thiaminechloride hydrochloride, riboflavin, pyridoxine hydrochloride, folicacid, manganese sulfate, potassium iodide, phylloquinone, biotin, sodiumselenite, vitamin D₃ and cyanocobalamin

E. Similac® Infant Formula

Usage: When an infant formula is needed: if the decision is made todiscontinue breastfeeding before age 1 year, if a supplement tobreastfeeding is needed or as a routine feeding if breastfeeding is notadopted.

Features:

Protein of appropriate quality and quantity for good growth;heat-denatured, which reduces the risk of milk-associated enteric bloodloss.

Fat from a blend of vegetable oils (doubly homogenized), providingessential linoleic acid that is easily absorbed.

Carbohydrate as lactose in proportion similar to that of human milk.

Low renal solute load to minimize stress on developing organs.

Powder, Concentrated Liquid and Ready To Feed forms.

Ingredients: (^({circle around (ll)})-D) Water, nonfat milk, lactose,soy oil, coconut oil, mono- and diglycerides, soy lecithin, abscorbicacid, carrageenan, choline chloride, taurine, m-inositol,alpha-tocopheryl acetate, zinc sulfate, niacinamid, ferrous sulfate,calcium pantothenate, cupric sulfate, vitamin A palmitate, thiaminechloride hydrochloride, riboflavin, pyridoxine hydrochloride, folicacid, manganese sulfate, phylloquinone, biotin, sodium selenite, vitaminD₃ and cyanocobalamin

F. Similac® NeoCare Premature Infant Formula with Iron

Usage: For premature infants' special nutritional needs after hospitaldischarge. Similac NeoCare is a nutritionally complete formula developedto provide premature infants with extra calories, protein, vitamins andminerals needed to promote catch-up growth and support development.

Features:

Reduces the need for caloric and vitamin supplementation. More calories(22 Cal/fl oz) then standard term formulas (20 Cal/fl oz).

Highly absorbed fat blend, with medium-chain triglycerides (MCT oil) tohelp meet the special digestive needs of premature infants.

Higher levels of protein, vitamins and minerals per 100 Calories toextend the nutritional support initiated in-hospital.

More calcium and phosphorus for improved bone mineralization.

Ingredients: ^({circle around (ll)})-D Corn syrup solids, nonfat milk,lactose, whey protein concentrate, soy oil, high-oleic safflower oil,fractionated coconut oil (medium-chain triglycerides), coconut oil,potassium citrate, calcium phosphate tribasic, calcium carbonate,ascorbic acid, magnesium chloride, potassium chloride, sodium chloride,taurine, ferrous sulfate, m-inositol, choline chloride, ascorbylpalmitate, L-carnitine, alpha-tocopheryl acetate, zinc sulfate,niacinamide, mixed tocopherols, sodium citrate, calcium pantothenate,cupric sulfate, thiamine chloride hydrochloride, vitamin A palmitate,beta carotene, riboflavin, pyridoxine hydrochloride, folic acid,manganese sulfate, phylloquinone, biotin, sodium selenite, vitamin D₃and cyanocobalamin.

G. Similac Natural Care Low-Iron Human Milk Fortifier Ready to Use, 24Cal/fl oz.

Usage: Designed to be mixed with human milk or to be fed alternativelywith human milk to low-birth-weight infants.

Ingredients: ^({circle around (ll)})-D Water, nonfat milk, hydrolyzedcornstarch, lactose, fractionated coconut oil (medium-chaintriglycerides), whey protein concentrate, soil oil, coconut oil, calciumphosphate tribasic, potassium citrate, magnesium chloride, sodiumcitrate, ascorbic acid, calcium carbonate, mono- and diglycerides, soylecithin, carrageenan, choline chloride, m-inositol, taurine,niacinamide, L-carnitine, alpha tocopheryl acetate, zinc sulfate,potassium chloride, calcium pantothenate, ferrous sulfate, cupricsulfate, riboflavin, vitamin A palmitate, thiamine chloridehydrochloride, pyridoxine hydrochloride, biotin, folic acid, manganesesulfate, phylloquinone, vitamin D₃, sodium selenite and cyanocobalamin.

Various PUFAs of this invention can be substituted and/or added to theinfant formulae described above and to other infant formulae known tothose in the art.

II. Nutritional Formulations

A. ENSURE®

Usage: ENSURE is a low-residue liquid food designed primarily as an oralnutritional supplement to be used with or between meals or, inappropriate amounts, as a meal replacement. ENSURE is lactose- andgluten-free, and is suitable for use in modified diets, includinglow-cholesterol diets. Although it is primarily an oral supplement, itcan be fed by tube.

Patient Conditions

For patients on modified diets

For elderly patients at nutrition risk

For patients with involuntary weight loss

For patients recovering from illness or surgery

For patients who need a low-residue diet

Ingredients

^({circle around (ll)})-D Water, Sugar (Sucrose), Maltodextrin (Corn),Calcium and Sodium Caseinates, High-Oleic Safflower Oil, Soy ProteinIsolate, Soy Oil, Canola Oil, Potassium Citrate, Calcium PhosphateTribasic, Sodium Citrate, Magnesium Chloride, Magnesium PhosphateDibasic, Artificial Flavor, Sodium Chloride, Soy Lecithin, CholineChloride, Ascorbic Acid, Carrageenan, Zinc Sulfate, Ferrous Sulfate,Alpha-Tocopheryl Acetate, Gellan Gum, Niacinamide, Calcium Pantothenate,Manganese Sulfate, Cupric Sulfate, Vitamin A Palmitate, ThiamineChloride Hydrochloride, Pyridoxine Hydrochloride, Riboflavin, FolicAcid, Sodium Molybdate, Chromium Chloride, Biotin, Potassium Iodide,Sodium Selenate.

B. ENSURE® BARS

Usage: ENSURE BARS are complete, balanced nutrition for supplemental usebetween or with meals. They provide a delicious, nutrient-richalternative to other snacks. ENSURE BARS contain <1 g lactose/bar, andChocolate Fudge Brownie flavor is gluten-free. (Honey Graham CrunchFlavor Contains Gluten.)

Patient Conditions

For patients who need extra calories, protein, vitamins and minerals

Especially useful for people who do not take in enough calories andnutrients

For people who have the ability to chew and swallow

Not to be used by anyone with a peanut allergy or any type of allergy tonuts.

Ingredients

Honey Graham Crunch—High-Fructose Corn Syrup, Soy Protein Isolate, BrownSugar, Honey, Maltodextrin (Corn), Crisp Rice (Milled Rice, Sugar[Sucrose], Salt [Sodium Chloride] and Malt), Oat Bran, PartiallyHydrogenated Cottonseed and Soy Oils, Soy Polysaccharide, Glycerine,Whey Protein Concentrate, Polydextrose, Fructose, Calcium Caseinate,Cocoa Powder, Artificial Flavors, Canola Oil, High-Oleic Safflower Oil,Nonfat Dry Milk, Whey Powder, Soy Lecithin and Corn Oil. Manufactured ina facility that processes nuts.

Vitamins and Minerals

Calcium Phosphate Tribasic, Potassium Phosphate Dibasic, MagnesiumOxide, Salt (Sodium Chloride), Potassium Chloride, Ascorbic Acid, FerricOrthophosphate, Alpha-Tocopheryl Acetate, Niacinamide, Zinc Oxide,Calcium Pantothenate, Copper Gluconate, Manganese Sulfate, Riboflavin,Beta-Carotene, Pyridoxine Hydrochloride, Thiamine Mononitrate, FolicAcid, Biotin, Chromium Chloride, Potassium Iodide, Sodium Selenate,Sodium Molybdate, Phylloquinone, Vitamin D₃ and Cyanocobalamin.

Protein

Honey Graham Crunch—The protein source is a blend of soy protein isolateand milk proteins. Soy protein isolate 74% Milk proteins 26%

Fat

Honey Graham Crunch—The fat source is a blend of partially hydrogenatedcottonseed and soybean, canola, high oleic safflower, and corn oils, andsoy lecithin. Partially hydrogenated 76% cottonseed and soybean oilCanola oil  8% High-oleic safflower oil  8% Corn oil  4% Soy lecithin 4%

Carbohydrate

Honey Graham Crunch—The carbohydrate source is a combination ofhigh-fructose corn syrup, brown sugar, maltodextrin, honey, crisp rice,glycerine, soy polysaccharide, and oat bran. High-fructose corn syrup24% Brown sugar 21% Maltodextrin 12% Honey 11% Crisp rice  9% Glycerine 9% Soy polysaccharide  7% Oat bran   7%\

C. ENSURE® HIGH PROTEIN

Usage: ENSURE HIGH PROTEIN is a concentrated, high-protein liquid fooddesigned for people who require additional calories, protein, vitamins,and minerals in their diets. It can be used as an oral nutritionalsupplement with or between meals or, in appropriate amounts, as a mealreplacement. ENSURE HIGH PROTEIN is lactose- and gluten-free, and issuitable for use by people recovering from general surgery or hipfractures and by patients at risk for pressure ulcers.

Patient Conditions

For patients who require additional calories, protein, vitamins, andminerals, such as patients recovering from general surgery or hipfractures, patients at risk for pressure ulcers, and patients onlow-cholesterol diets

Features

Low in saturated fat

Contains 6 g of total fat and <5 mg of cholesterol per serving

Rich, creamy taste

Excellent source of protein, calcium, and other essential vitamins andminerals

For low-cholesterol diets

Lactose-free, easily digested

Ingredients

Vanilla Supreme: ^({circle around (ll)})-D Water, Sugar (Sucrose),Maltodextrin (Corn), Calcium and Sodium Caseinates, High-Oleic SafflowerOil, Soy Protein Isolate, Soy Oil, Canola Oil, Potassium Citrate,Calcium Phosphate Tribasic, Sodium Citrate, Magnesium Chloride,Magnesium Phosphate Dibasic, Artificial Flavor, Sodium Chloride, SoyLecithin, Choline Chloride, Ascorbic Acid, Carrageenan, Zinc Sulfate,Ferrous Sulfate, Alpha-Tocopheryl Acetate, Gellan Gum, Niacinamide,Calcium Pantothenate, Manganese Sulfate, Cupric Sulfate, Vitamin APalmitate, Thiamine Chloride Hydrochloride, Pyridoxine Hydrochloride,Riboflavin, Folio Acid, Sodium Molybdate, Chromium Chloride, Biotin,Potassium Iodide, Sodium Selenate, Phylloquinone, Vitamin D.3 andCyanocobalarnin.

Protein

The protein source is a blend of two high-biologic-value proteins:casein and soy. Sodium and calcium caseinates 85% Soy protein isolatel15%

Fat

The fat source is a blend of three oils: high-oleic safflower, canola,and soy. High-oleic safflower oil 40% Canola oil 30% Soy oil 30%

The level of fat in ENSURE HIGH PROTEIN meets American Heart Association(AHA) guidelines. The 6 grams of fat in ENSURE HIGH PROTEIN represent24% of the total calories, with 2.6% of the fat being from saturatedfatty acids and 7.9% from polyunsaturated fatty acids. These values arewithin the AHA guidelines of ≦30% of total calories from fat, <10% ofthe calories from saturated fatty acids, and ≦10% of total calories frompolyunsaturated fatty acids.

Carbohydrate

ENSURE HIGH PROTEIN contains a combination of maltodextrin and sucrose.The mild sweetness and flavor variety (vanilla supreme, chocolate royal,wild berry, and banana), plus VARI-FLAVORSO® Flavor Pacs in pecan,cherry, strawberry, lemon, and orange, help to prevent flavor fatigueand aid in patient compliance.

Vanilla and Other Nonchocolate Flavors

Sucrose 60% Maltodextrin 40%

Chocolate

Sucrose 70% Maltodextrin 30%

D. ENSURE® LIGHT

Usage: ENSURE LIGHT is a low-fat liquid food designed for use as an oralnutritional supplement with or between meals. ENSURE LIGHT is lactose-and gluten-free, and is suitable for use in modified diets, includinglow-cholesterol diets.

Patient Conditions

For normal-weight or overweight patients who need extra nutrition in asupplement that contains 50% less fat and 20% fewer calories than ENSURE

For healthy adults who don't eat right and need extra nutrition

Features

Low in fat and saturated fat

Contains 3 g of total fat per serving and <5 mg cholesterol

Rich, creamy taste

Excellent source of calcium and other essential vitamins and minerals

For low-cholesterol diets

Lactose-free, easily digested

Ingredients

French Vanilla: ^({circle around (ll)})-D Water, Maltodextrin (Corn),Sugar (Sucrose), Calcium Caseinate, High-Oleic Safflower Oil, CanolaOil, Magnesium Chloride, Sodium Citrate, Potassium Citrate, PotassiumPhosphate Dibasic, Magnesium Phosphate Dibasic, Natural and ArtificialFlavor, Calcium Phosphate Tribasic, Cellulose Gel, Choline Chloride, SoyLecithin, Carrageenan, Salt (Sodium Chloride), Ascorbic Acid, CelluloseGum, Ferrous Sulfate, Alpha-Tocopheryl Acetate, Zinc Sulfate,Niacinamide, Manganese Sulfate, Calcium Pantothenate, Cupric Sulfate,Thiamine Chloride Hydrochloride, Vitamin A Palmitate, PyridoxineHydrochloride, Riboflavin, Chromium Chloride, Folic Acid, SodiumMolybdate, Biotin, Potassium Iodide, Sodium Selenate, Phylloquinone,Vitamin D₃ and Cyanocobalamin.

Protein

The protein source is calcium caseinate. Calcium caseinate 100%

Fat

The fat source is a blend of two oils: high-oleic safflower and canola.High-oleic safflower oil 70% Canola oi 30%

The level of fat in ENSURE LIGHT meets American Heart Association (AHA)guidelines. The 3 grams of fat in ENSURE LIGHT represent 13.5% of thetotal calories, with 1.4% of the fat being from saturated fatty acidsand 2.6% from polyunsaturated fatty acids. These values are within theAHA guidelines of ≦30% of total calories from fat, <10% of the caloriesfrom saturated fatty acids, and ≦10% of total calories frompolyunsaturated fatty acids.

Carbohydrate

ENSURE LIGHT contains a combination of maltodextrin and sucrose. Thechocolate flavor contains corn syrup as well. The mild sweetness andflavor variety (French vanilla, chocolate supreme, strawberry swirl),plus VARI-FLAVORS® Flavor Pacs in pecan, cherry, strawberry, lemon, andorange, help to prevent flavor fatigue and aid in patient compliance.

Vanilla and Other Nonchocolate Flavors

Sucrose 51% Maltodextrin 49%

Chocolate

Sucrose 47.0% Corn Syrup 26.5% Maltodextrin 26.5%

Vitamins and Minerals

An 8-fl-oz serving of ENSURE LIGHT provides at least 25% of the RDIs for24 key vitamins and minerals.

Caffeine

Chocolate flavor contains 2.1 mg caffeine/8 fl oz.

E. ENSURE PLUS®

Usage: ENSURE PLUS is a high-calorie, low-residue liquid food for usewhen extra calories and nutrients, but a normal concentration ofprotein, are needed. It is designed primarily as an oral nutritionalsupplement to be used with or between meals or, in appropriate amounts,as a meal replacement. ENSURE PLUS is lactose- and gluten-free. Althoughit is primarily an oral nutritional supplement, it can be fed by tube.

Patient Conditions

For patients who require extra calories and nutrients, but a normalconcentration of protein, in a limited volume

For patients who need to gain or maintain healthy weight

Features

Rich, creamy taste

Good source of essential vitamins and minerals

Ingredients

Vanilla: ^({circle around (ll)})-D Water, Corn Syrup, Maltodextrin(Corn), Corn Oil, Sodium and Calcium Caseinates, Sugar (Sucrose), SoyProtein Isolate, Magnesium Chloride, Potassium Citrate, CalciumPhosphate Tribasic, Soy Lecithin, Natural and Artificial Flavor, SodiumCitrate, Potassium Chloride, Choline Chloride, Ascorbic Acid,Carrageenan, Zinc Sulfate, Ferrous Sulfate, Alpha-Tocopheryl Acetate,Niacinamide, Calcium Pantothenate, Manganese Sulfate, Cupric Sulfate,Thiamine Chloride Hydrochloride, Pyridoxine Hydrochloride, Riboflavin,Vitamin A Palmitate, Folic Acid, Biotin, Chromium Chloride, SodiumMolybdate, Potassium Iodide, Sodium Selenite, Phylloquinone,Cyanocobalamin and Vitamin D₃.

Protein

The protein source is a blend of two high-biologic-value proteins:casein and soy. Sodium and calcium caseinates 84% Soy protein isolate16%

Fat

The fat source is corn oil. Corn oil 100%

Carbohydrate

ENSURE PLUS contains a combination of maltodextrin and sucrose. The mildsweetness and flavor variety (vanilla, chocolate, strawberry, coffee,buffer pecan, and eggnog), plus VARI-FLAVORS® Flavor Pacs in pecan,cherry, strawberry, lemon, and orange, help to prevent flavor fatigueand aid in patient compliance.

Vanilla, Strawberry, Butter Pecan, and Coffee Flavors

Corn Syrup 39% Maltodextrin 38% Sucrose 23%

Chocolate and Eggnog Flavors

Corn Syrup 36% Maltodextrin 34% Sucrose 30%

Vitamins and Minerals

An 8-fl-oz serving of ENSURE PLUS provides at least 15% of the RDIs for25 key Vitamins and minerals.

Caffeine

Chocolate flavor contains 3.1 mg Caffeine/8 fl oz. Coffee flavorcontains a trace amount of caffeine.

F. ENSURE PLUS® HN

Usage: ENSURE PLUS HN is a nutritionally complete high-calorie,high-nitrogen liquid food designed for people with higher calorie andprotein needs or limited volume tolerance. It may be used for oralsupplementation or for total nutritional support by tube. ENSURE PLUS HNis lactose- and gluten-free.

Patient Conditions

For patients with increased calorie and protein needs, such as followingsurgery or injury

For patients with limited volume tolerance and early satiety

Features

For supplemental or total nutrition

For oral or tube feeding

1.5 CaVmL

High nitrogen

Calorically dense

Ingredients

Vanilla: ^({circle around (ll)})-D Water, Maltodextrin (Corn), Sodiumand Calcium Caseinates, Corn Oil, Sugar (Sucrose), Soy Protein Isolate,Magnesium Chloride, Potassium Citrate, Calcium Phosphate Tribasic, SoyLecithin, Natural and Artificial Flavor, Sodium Citrate, CholineChloride, Ascorbic Acid, Taurine, L-Carnitine, Zinc Sulfate, FerrousSulfate, Alpha-Tocopheryl Acetate, Niacinamide, Carrageenan, CalciumPantothenate, Manganese Sulfate, Cupric Sulfate, Thiamine ChlorideHydrochloride, Pyridoxine Hydrochloride, Riboflavin, Vitamin APalmitate, Folic Acid, Biotin, Chromium Chloride, Sodium Molybdate,Potassium Iodide, Sodium Selenite, Phylloquinone, Cyanocobalamin andVitamin D₃.

G. ENSURE® POWDER

Usage: ENSURE POWDER (reconstituted with water) is a low-residue liquidfood designed primarily as an oral nutritional supplement to be usedwith or between meals. ENSURE POWDER is lactose- and gluten-free, and issuitable for use in modified diets, including low-cholesterol diets.

Patient Conditions

For patients on modified diets

For elderly patients at nutrition risk

For patients recovering from illness/surgery

For patients who need a low-residue diet

Features

Convenient, easy to mix

Low in saturated fat

Contains 9 g of total fat and <5 mg of cholesterol per serving

High in vitamins and minerals

For low-cholesterol diets

Lactose-free, easily digested

Ingredients: ^({circle around (ll)})-D Corn Syrup, Maltodextrin (Corn),Sugar (Sucrose), Corn Oil, Sodium and Calcium Caseinates, Soy ProteinIsolate, Artificial Flavor, Potassium Citrate, Magnesium Chloride,Sodium Citrate, Calcium Phosphate Tribasic, Potassium Chloride, SoyLecithin, Ascorbic Acid, Choline Chloride, Zinc Sulfate, FerrousSulfate, Alpha-Tocopheryl Acetate, Niacinamide, Calcium Pantothenate,Manganese Sulfate, Thiamine Chloride Hydrochloride, Cupric Sulfate,Pyridoxine Hydrochloride, Riboflavin, Vitamin A Palmitate, Folic Acid,Biotin, Sodium Molybdate, Chromium Chloride, Potassium Iodide, SodiumSelenate, Phylloquinone, Vitamin D₃ and Cyanocobalamin.

Protein

The protein source is a blend of two high-biologic-value proteins:casein and soy. Sodium and calcium caseinates 84% Soy protein isolate16%

Fat

The fat source is corn oil. The fat source is corn oil. Corn oil 100%

Carbohydrate

ENSURE POWDER contains a combination of corn syrup, maltodextrin, andsucrose. The mild sweetness of ENSURE POWDER, plus VARI-FLAVORS® FlavorPacs in pecan, cherry, strawberry, lemon, and orange, helps to preventflavor fatigue and aid in patient compliance.

Vanilla

Corn Syrup 35% Maltodextrin 35% Sucrose 30%

H. ENSURE® PUDDING

Usage: ENSURE PUDDING is a nutrient-dense supplement providing balancednutrition in a nonliquid form to be used with or between meals. It isappropriate for consistency-modified diets (e.g., soft, pureed, or fullliquid) or for people with swallowing impairments. ENSURE PUDDING isgluten-free.

Patient Conditions

For patients on consistency-modified diets (e.g., soft, pureed, or fullliquid)

For patients with swallowing impairments

Features

Rich and creamy, good taste

Good source of essential vitamins and minerals Convenient—needs norefrigeration

Gluten-free

Nutrient Profile per 5 oz: Calories 250, Protein 10.9%, Total Fat 34.9%,Carbohydrate 54.2%

Ingredients

Vanilla: ^({circle around (ll)})-D Nonfat Milk, Water, Sugar (Sucrose),Partially Hydrogenated Soybean Oil, Modified Food Starch, MagnesiumSulfate. Sodium Stearoyl Lactylate, Sodium Phosphate Dibasic, ArtificialFlavor, Ascorbic Acid, Zinc Sulfate, Ferrous Sulfate, Alpha-TocopherylAcetate, Choline Chloride, Niacinamide, Manganese Sulfate, CalciumPantothenate, FD&C Yellow #5, Potassium Citrate, Cupric Sulfate, VitaminA Palmitate, Thiamine Chloride Hydrochloride, Pyridoxine Hydrochloride,Riboflavin, FD&C Yellow #6, Folic Acid, Biotin, Phylloquinone, VitaminD₃ and Cyanocobalamin.

Protein

The protein source is nonfat milk. Nonfat milk 100%

Fat

The fat source is hydrogenated soybean oil. Hydrogenated soybean oil100%

Carbohydrate

ENSURE PUDDING contains a combination of sucrose and modified foodstarch. The mild sweetness and flavor variety (vanilla, chocolate,butterscotch, and tapioca) help prevent flavor fatigue. The productcontains 9.2 grams of lactose per serving.

Vanilla and Other Nonchocolate Flavors

Sucrose 56% Lactose 27% Modified food starch 17%

Chocolate

Sucrose 58% Lactose 26% Modified food starch 16%

I. ENSURE® WITH FIBER

Usage: ENSURE WITH FIBER is a fiber-containing, nutritionally completeliquid food designed for people who can benefit from increased dietaryfiber and nutrients. ENSURE WITH FIBER is suitable for people who do notrequire a low-residue diet. It can be fed orally or by tube, and can beused as a nutritional supplement to a regular diet or, in appropriateamounts, as a meal replacement. ENSURE WITH FIBER is lactose- andgluten-free, and is suitable for use in modified diets, includinglow-cholesterol diets.

Patient Conditions

For patients who can benefit from increased dietary fiber and nutrients

Features

New advanced formula-low in saturated fat, higher in vitamins andminerals

Contains 6 g of total fat and <5 mg of cholesterol per serving

Rich, creamy taste

Good source of fiber

Excellent source of essential vitamins and minerals

For low-cholesterol diets

Lactose- and gluten-free

Ingredients

Vanilla: ^({circle around (ll)})-D Water, Maltodextrin (Corn), Sugar(Sucrose), Sodium and Calcium Caseinates, Oat Fiber, High-OleicSafflower Oil, Canola Oil, Soy Protein Isolate, Corn Oil, Soy Fiber,Calcium Phosphate Tribasic, Magnesium Chloride, Potassium Citrate,Cellulose Gel, Soy Lecithin, Potassium Phosphate Dibasic, SodiumCitrate, Natural and Artificial Flavors, Choline Chloride, MagnesiumPhosphate, Ascorbic Acid, Cellulose Gum, Potassium Chloride,Carrageenan, Ferrous Sulfate, Alpha-Tocopheryl Acetate, Zinc Sulfate,Niacinamide, Manganese Sulfate, Calcium Pantothenate, Cupric Sulfate,Vitamin A Palmitate, Thiamine Chloride Hydrochloride, PyridoxineHydrochloride, Riboflavin, Folic Acid, Chromium Chloride, Biotin, SodiumMolybdate, Potassium Iodide, Sodium Selenate, Phylloquinone, Vitamin D₃and Cyanocobalamin.

Protein

The protein source is a blend of two high-biologic-value proteins—caseinand soy. Sodium and calcium caseinates 80% Soy protein isolate 20%

Fat

The fat source is a blend of three oils: high-oleic safflower, canola,and corn. High-oleic safflower oil 40% Canola oil 40% Corn oil 20%

The level of fat in ENSURE WITH FIBER meets American Heart Association(AHA) guidelines. The 6 grams of fat in ENSURE WITH FIBER represent 22%of the total calories, with 2.01% of the fat being from saturated fattyacids and 6.7% from polyunsaturated fatty acids. These values are withinthe AHA guidelines of ≦30% of total calories from fat, <10% of thecalories from saturated fatty acids, and ≦10% of total calories frompolyunsaturated fatty acids.

Carbohydrate

ENSURE WITH FIBER contains a combination of maltodextrin and sucrose.The mild sweetness and flavor variety (vanilla, chocolate, and butterpecan), plus VARI-FLAVORS® Flavor Pacs in pecan, cherry, strawberry,lemon, and orange, help to prevent flavor fatigue and aid in patientcompliance.

Vanilla and Other Nonchocolate Flavors

Maltodextrin 66% Sucrose 25% Oat Fiber  7% Soy Fiber  2%

Chocolate

Maltodextrin 55% Sucrose 36% Oat Fiber  7% Soy Fiber  2%

Fiber

The fiber blend used in ENSURE WITH FIBER consists of oat fiber and soypolysaccharide. This blend results in approximately 4 grams of totaldietary fiber per 8-fl-oz can. The ratio of insoluble to soluble fiberis 95:5.

The various nutritional supplements described above and known to othersof skill in the art can be substituted and/or supplemented with thePUFAs of this invention.

J. Oxepa™ Nutritional Product

Oxepa is low-carbohydrate, calorically dense enteral nutritional productdesigned for the dietary management of patients with or at risk forARDS. It has a unique combination of ingredients, including a patentedoil blend containing eicosapentaenoic acid (EPA from fish oil),γ-linolenic acid (GLA from borage oil), and elevated antioxidant levels.

Caloric Distribution

Caloric density is high at 1.5 Cal/mL (355 Cal/8 fl oz), to minimize thevolume required to meet energy needs.

The distribution of Calories in Oxepa is shown in Table 7. TABLE 7Caloric Distribution of Oxepa per 8 fl oz. per liter % of Cal Calories355 1,500 — Fat (g) 22.2 93.7 55.2 Carbohydrate (g) 25 105.5 28.1Protein (g) 14.8 62.5 16.7 Water (g) 186 785 —

Fat

Oxepa contains 22.2 g of fat per 8-fl oz serving (93.7 g/L).

The fat source is a oil blend of 31.8% canola oil, 25% medium-chaintriglycerides (MCTs), 20% borage oil, 20% fish oil, and 3.2% soylecithin. The typical fatty acid profile of Oxepa is shown in Table 8.

Oxepa provides a balanced amount of polyunsaturated, monounsaturated,and saturated fatty acids, as shown in Table 10.

Medium-chain triglycerides (MCTs)—25% of the fat blend—aid gastricemptying because they are absorbed by the intestinal tract withoutemulsification by bile acids.

The various fatty acid components of Oxepa™ nutritional product can besubstituted and/or supplemented with the PUFAs of this invention. TABLE8 Typical Fatty Acid Profile % Total Fatty Acids g/8 fl oz* g/L* Caproic(6:0 0.2 0.04 0.18 Caprylic (8:0) 14.69 3.1 13.07 Capric (10:0) 11.062.33 9.87 Palmitic (16:0) 5.59 1.18 4.98 Palmitoleic (16:1n-7) 1.82 0.381.62 Stearic (18:0) 1.84 0.39 1.64 Oleic (18:ln-9) 24.44 5.16 21.75Linoleic (18:2n-6) 16.28 3.44 14.49 α-Linolenic (18:3n-3) 3.47 0.73 3.09γ-Linolenic (18:3n-6) 4.82 1.02 4.29 Eicosapentaenoic 5.11 1.08 4.55(20:5n-3) n-3-Docosapentaenoic 0.55 0.12 0.49 (22:Sn-3) Docosahexaenoic2.27 0.48 2.02 (22:6n-3) Others 7.55 1.52 6.72*Fatty acids equal approximately 95% of total fat.

TABLE 9 Fat Profile of Oxepa. % of total calories from fat 55.2Polyunsaturated fatty acids 31.44 g/L Monounsaturated fatty acids 25.53g/L Saturated fatty acids 32.38 gIL n-6 to n-3 ratio 1.75:1 Cholesterol9.49 mg/8 fl oz 40.1 mg/L

Carbohydrate

The carbohydrate content is 25.0 g per 8-fl-oz serving (105.5 g/L).

The carbohydrate sources are 45% maltodextrin (a complex carbohydrate)and 55% sucrose (a simple sugar), both of which are readily digested andabsorbed.

The high-fat and low-carbohydrate content of Oxepa is designed tominimize carbon dioxide (CO₂) production. High CO₂ levels can complicateweaning in ventilator-dependent patients. The low level of carbohydratealso may be useful for those patients who have developed stress-inducedhyperglycemia.

Oxepa is lactose-free.

Dietary carbohydrate, the amino acids from protein, and the glycerolmoiety of fats can be converted to glucose within the body. Throughoutthis process, the carbohydrate requirements of glucose-dependent tissues(such as the central nervous system and red blood cells) are met.However, a diet free of carbohydrates can lead to ketosis, excessivecatabolism of tissue protein, and loss of fluid and electrolytes. Theseeffects can be prevented by daily ingestion of 50 to 100 g of digestiblecarbohydrate, if caloric intake is adequate. The carbohydrate level inOxepa is also sufficient to minimize gluconeogenesis, if energy needsare being met.

Protein

Oxepa contains 14.8 g of protein per 8-fl-oz serving (62.5 g/L).

The total calorie/nitrogen ratio (150:1) meets the need of stressedpatients.

Oxepa provides enough protein to promote anabolism and the maintenanceof lean body mass without precipitating respiratory problems. Highprotein intakes are a concern in patients with respiratoryinsufficiency. Although protein has little effect on CO₂ production, ahigh protein diet will increase ventilatory drive.

The protein sources of Oxepa are 86.8% sodium caseinate and 13.2%calcium caseinate.

As demonstrated in Table 11, the amino acid profile of the proteinsystem in Oxepa meets or surpasses the standard for high quality proteinset by the National Academy of Sciences.

Oxepa is gluten-free.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

1-98. (canceled)
 99. A nutritional formula comprising a microbial oil or fraction thereof obtained according to a method for obtaining altered long chain polyunsaturated fatty acid biosynthesis, the method comprising: growing a plant having cells which contain one or more transgenes, derived from a fungus or algae, which encodes a transgene expression product which desaturates a fatty acid molecule at a carbon selected from the group consisting of carbon 6 and carbon 12 from the carboxyl end of said fatty acid molecule, wherein said one or more transgenes is operably associated with an expression control sequence, under conditions whereby said one or more transgenes is expressed, whereby long chain polyunsaturated fatty acid biosynthesis in said cells is altered.
 100. The nutritional formula of claim 99, wherein said nutritional formula is selected from the group consisting of an infant formula, a dietary supplement, and a dietary substitute.
 101. The nutritional formula of claim 100, wherein said infant formula, dietary supplement or dietary supplement is in the form of a liquid or a solid.
 102. The nutritional formula of claim 100, wherein said nutritional formula is an infant formula, and wherein said infant formula further comprises at least one macronutrient selected from the group consisting of coconut oil, soy oil, canola oil, mono- and diglycerides, glucose, edible lactose, electrodialysed whey, electrodialysed skim milk, milk whey, soy protein, and other protein hydrolysates.
 103. The nutritional formula of claim 102 further comprising at least one vitamin selected from the group consisting of Vitamins A, C, D, E, and B complex; and at least one mineral selected from the group consisting of calcium, magnesium, zinc, manganese, sodium, potassium, phosphorus, copper, chloride, iodine, selenium, and iron.
 104. The nutritional formula of claim 100, wherein said nutritional formula is a dietary supplement, and wherein said dietary supplement further comprises at least one macronutrient selected from the group consisting of coconut oil, soy oil, canola oil, mono- and diglycerides, glucose, edible lactose, electrodialysed whey, electrodialysed skim milk, milk whey, soy protein, and other protein hydrolysates.
 105. The nutritional formula of claim 104 further comprising at least one vitamin selected from the group consisting of Vitamins A, C, D, E, and B complex; and at least one mineral selected from the group consisting of calcium, magnesium, zinc, manganese, sodium, potassium, phosphorus, copper, chloride, iodine, selenium, and iron.
 106. The nutritional formula of claim 104 wherein said dietary supplement is administered to a human or an animal.
 107. The nutritional formula of claim 100, wherein said nutritional formula is a dietary substitute, and wherein said dietary substitute further comprises at least one macronutrient selected from the group consisting of coconut oil, soy oil, canola oil, mono- and diglycerides, glucose, edible lactose, electrodialysed whey, electrodialysed skim milk, milk whey, soy protein, and other protein hydrolysates.
 108. The nutritional formula of claim 107 further comprising at least one vitamin selected from the group consisting of Vitamins A, C, D, E, and B complex; and at least one mineral selected from the group consisting of calcium, magnesium, zinc, manganese, sodium, potassium, phosphorus, copper, chloride, iodine, selenium, and iron.
 109. The nutritional formula of claim 107, wherein said dietary substitute is administered to a human or animal.
 110. A nutritional formula comprising a microbial oil or fraction thereof produced by: growing one or more transgenic microbial cells under suitable conditions whereby said cells express a transgenic polypeptide wherein the sequence of said polypeptide comprises a sequence selected from the group consisting of residues 50-53, 39-43, 172-176, 204-213, and 390-402 of SEQ ID NO:2.
 111. The nutritional formula of claim 110, wherein said nutritional formula is selected from the group consisting of an infant formula, a dietary supplement, and a dietary substitute.
 112. The nutritional formula of claim 111, wherein said infant formula, dietary supplement or dietary supplement is in the form of a liquid or a solid.
 113. The nutritional formula of claim 111, wherein said nutritional formula is an infant formula, and wherein said infant formula further comprises at least one macronutrient selected from the group consisting of coconut oil, soy oil, canola oil, mono- and diglycerides, glucose, edible lactose, electrodialysed whey, electrodialysed skim milk, milk whey, soy protein, and other protein hydrolysates.
 114. The nutritional formula of claim 113 further comprising at least one vitamin selected from the group consisting of Vitamins A, C, D, E, and B complex; and at least one mineral selected from the group consisting of calcium, magnesium, zinc, manganese, sodium, potassium, phosphorus, copper, chloride, iodine, selenium, and iron.
 115. The nutritional formula of claim 111, wherein said nutritional formula is a dietary supplement, and wherein said dietary supplement further comprises at least one macronutrient selected from the group consisting of coconut oil, soy oil, canola oil, mono- and diglycerides, glucose, edible lactose, electrodialysed whey, electrodialysed skim milk, milk whey, soy protein, and other protein hydrolysates.
 116. The nutritional formula of claim 115 further comprising at least one vitamin selected from the group consisting of Vitamins A, C, D, E, and B complex; and at least one mineral selected from the group consisting of calcium, magnesium, zinc, manganese, sodium, potassium, phosphorus, copper, chloride, iodine, selenium, and iron.
 117. The nutritional formula of claim 115 wherein said dietary supplement is administered to a human or an animal.
 118. The nutritional formula of claim 111, wherein said nutritional formula is a dietary substitute, and wherein said dietary substitute further comprises at least one macronutrient selected from the group consisting of coconut oil, soy oil, canola oil, mono- and diglycerides, glucose, edible lactose, electrodialysed whey, electrodialysed skim milk, milk whey, soy protein, and other protein hydrolysates.
 119. The nutritional formula of claim 118 further comprising at least one vitamin selected from the group consisting of Vitamins A, C, D, E, and B complex; and at least one mineral selected from the group consisting of calcium, magnesium, zinc, manganese, sodium, potassium, phosphorus, copper, chloride, iodine, selenium, and iron.
 120. The nutritional formula of claim 118, wherein said dietary substitute is administered to a human or animal.
 121. A nutritional formula comprising a microbial oil or fraction thereof produced by: growing microbial cells which contain one or more transgenes which encode a transgene expression product under conditions whereby said one or more transgenes are expressed, whereby long chain polyunsaturated fatty acid biosynthesis in said cells is altered whereby said transgene comprises a nucleotide sequence which encodes a polypeptide wherein the sequence of the polypeptide comprises a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26.
 122. The nutritional formula of claim 121, wherein said nutritional formula is selected from the group consisting of an infant formula, a dietary supplement, and a dietary substitute.
 123. The nutritional formula of claim 122, wherein said infant formula, dietary supplement or dietary supplement is in the form of a liquid or a solid.
 124. The nutritional formula of claim 122, wherein said nutritional formula is an infant formula, and wherein said infant formula further comprises at least one macronutrient selected from the group consisting of coconut oil, soy oil, canola oil, mono- and diglycerides, glucose, edible lactose, electrodialysed whey, electrodialysed skim milk, milk whey, soy protein, and other protein hydrolysates.
 125. The nutritional formula of claim 124 further comprising at least one vitamin selected from the group consisting of Vitamins A, C, D, E, and B complex; and at least one mineral selected from the group consisting of calcium, magnesium, zinc, manganese, sodium, potassium, phosphorus, copper, chloride, iodine, selenium, and iron.
 126. The nutritional formula of claim 122, wherein said nutritional formula is a dietary supplement, and wherein said dietary supplement further comprises at least one macronutrient selected from the group consisting of coconut oil, soy oil, canola oil, mono- and diglycerides, glucose, edible lactose, electrodialysed whey, electrodialysed skim milk, milk whey, soy protein, and other protein hydrolysates.
 127. The nutritional formula of claim 126 further comprising at least one vitamin selected from the group consisting of Vitamins A, C, D, E, and B complex; and at least one mineral selected from the group consisting of calcium, magnesium, zinc, manganese, sodium, potassium, phosphorus, copper, chloride, iodine, selenium, and iron.
 128. The nutritional formula of claim 126 wherein said dietary supplement is administered to a human or an animal.
 129. The nutritional formula of claim 122, wherein said nutritional formula is a dietary substitute, and wherein said dietary substitute further comprises at least one macronutrient selected from the group consisting of coconut oil, soy oil, canola oil, mono- and diglycerides, glucose, edible lactose, electrodialysed whey, electrodialysed skim milk, milk whey, soy protein, and other protein hydrolysates.
 130. The nutritional formula of claim 129 further comprising at least one vitamin selected from the group consisting of Vitamins A, C, D, E, and B complex; and at least one mineral selected from the group consisting of calcium, magnesium, zinc, manganese, sodium, potassium, phosphorus, copper, chloride, iodine, selenium, and iron.
 131. The nutritional formula of claim 129, wherein said dietary substitute is administered to a human or animal.
 132. A nutritional formula comprising a microbial oil or fraction thereof produced by: growing one or more transgenic microbial cells under suitable conditions whereby said cells express one or more transgenic polypeptides wherein the sequence of said one or more polypeptides comprises a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26.
 133. The nutritional formula of claim 132, wherein said nutritional formula is selected from the group consisting of an infant formula, a dietary supplement, and a dietary substitute.
 134. The nutritional formula of claim 133, wherein said infant formula, dietary supplement or dietary supplement is in the form of a liquid or a solid.
 135. The nutritional formula of claim 133, wherein said nutritional formula is an infant formula, and wherein said infant formula further comprises at least one macronutrient selected from the group consisting of coconut oil, soy oil, canola oil, mono- and diglycerides, glucose, edible lactose, electrodialysed whey, electrodialysed skim milk, milk whey, soy protein, and other protein hydrolysates.
 136. The nutritional formula of claim 135 further comprising at least one vitamin selected from the group consisting of Vitamins A, C, D, E, and B complex; and at least one mineral selected from the group consisting of calcium, magnesium, zinc, manganese, sodium, potassium, phosphorus, copper, chloride, iodine, selenium, and iron.
 137. The nutritional formula of claim 133, wherein said nutritional formula is a dietary supplement, and wherein said dietary supplement further comprises at least one macronutrient selected from the group consisting of coconut oil, soy oil, canola oil, mono- and diglycerides, glucose, edible lactose, electrodialysed whey, electrodialysed skim milk, milk whey, soy protein, and other protein hydrolysates.
 138. The nutritional formula of claim 137 further comprising at least one vitamin selected from the group consisting of Vitamins A, C, D, E, and B complex; and at least one mineral selected from the group consisting of calcium, magnesium, zinc, manganese, sodium, potassium, phosphorus, copper, chloride, iodine, selenium, and iron.
 139. The nutritional formula of claim 137 wherein said dietary supplement is administered to a human or an animal.
 140. The nutritional formula of claim 133, wherein said nutritional formula is a dietary substitute, and wherein said dietary substitute further comprises at least one macronutrient selected from the group consisting of coconut oil, soy oil, canola oil, mono- and diglycerides, glucose, edible lactose, electrodialysed whey, electrodialysed skim milk, milk whey, soy protein, and other protein hydrolysates.
 141. The nutritional formula of claim 140 further comprising at least one vitamin selected from the group consisting of Vitamins A, C, D, E, and B complex; and at least one mineral selected from the group consisting of calcium, magnesium, zinc, manganese, sodium, potassium, phosphorus, copper, chloride, iodine, selenium, and iron.
 142. The nutritional formula of claim 140, wherein said dietary substitute is administered to a human or animal. 